Nucleic acids for inhibiting expression of cnnm4 in a cell

ABSTRACT

The invention relates to nucleic acid products that interfere with or inhibit CNNM4 (Cyclin M4) gene expression. It further relates to therapeutic uses of CNNM4 inhibition for the treatment of diseases, such as liver diseases including non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), liver steatosis, liver fibrosis, liver cirrhosis, liver cancer and other diseases associated with magnesium dysregulation.

FIELD OF THE INVENTION

The invention relates to nucleic acid products that interfere with or inhibit CNNM4 (Cyclin M4) gene expression. It further relates to therapeutic uses of CNNM4 inhibition for the treatment of diseases, among others, of liver diseases such as non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), liver steatosis, liver fibrosis, liver cirrhosis, liver cancer and other diseases associated with magnesium dysregulation.

BACKGROUND

Double-stranded RNAs (dsRNA) able to bind through complementary base pairing to expressed mRNAs have been shown to block gene expression (Fire et al., 1998, Nature. 1998 Feb 19;391(6669):806-11 and Elbashir et al., 2001, Nature. 2001 May 24;411(6836):494-8) by a mechanism that has been termed “RNA interference (RNAi)”. Short dsRNAs direct gene specific, post transcriptional silencing in many organisms, including vertebrates, and have become a useful tool for studying gene function. RNAi is mediated by the RNA induced silencing complex (RISC), a sequence specific, multi component nuclease that degrades messenger RNAs having sufficient complementary or homology to the silencing trigger loaded into the RISC complex. Interfering RNAs such as siRNAs, antisense RNAs, and micro RNAs, are oligonucleotides that prevent the formation of proteins by gene silencing, i.e., inhibiting gene translation of the protein through degradation of mRNA molecules. Gene silencing agents are becoming increasingly important for therapeutic applications in medicine.

According to Watts and Corey in the Journal of Pathology (2012; Vol 226, p 365-379), there are algorithms that can be used to design nucleic acid silencing triggers, but all of these have severe limitations. It may take various experimental methods to identify potent siRNAs, as algorithms do not take into account factors such as tertiary structure of the target mRNA or the involvement of RNA binding proteins. Therefore, the discovery of a potent nucleic acid silencing trigger with minimal off-target effects is a complex process. For the pharmaceutical development of these highly charged molecules, it is necessary that they can be synthesised economically, distributed to target tissues, enter cells and function within acceptable limits of toxicity.

Chronic liver diseases comprise a group of hepatic pathologies of different aetiologies. Non-alcoholic fatty liver disease (NAFLD) has an incidence of 20-30% in the global population (Younossi et al, Hepatology 64, 73-84 (2016)) and includes a spectrum of hepatic disorders. It can progress from simple steatosis, the accumulation of lipid in the liver, to non-alcoholic steatohepatitis (NASH), which is characterized by steatosis with inflammation and occasional fibrosis. While steatosis is considered to be a relatively benign and reversible condition, there is a risk of progression to NASH, which is a chronic, progressive disease characterized by fatty liver and liver cell injury that can develop into fibrosis, cirrhosis and hepatocellular carcinoma (HCC) (Povsic et al., Adv. Ther. 36, 1574-1594 (2019)). About 20% of patients with NASH develop cirrhosis, an irreversible pathology characterized by extracellular matrix deposition that causes liver dysfunction. About a quarter of patients with liver fibrosis develop HCC, which is the most frequent form of liver cancer and the fifth cause of mortality and morbidity in the world (Younossi et al., Clin. Gastroenterol. Hepatol. 4,748-755 (2018)). Very few patients are eligible for currently available therapeutic intervention when diagnosed, and survival rates are poor, with on average of only 6-20 months survival after diagnosis. There is therefore a clear unmet need for treatment of these and similar chronic diseases.

The liver can also suffer from acute damages. This organ plays a central role in drug metabolism and clearance and drug-induced liver injury (DILI) can result from drug overdose. DILI is the main cause of acute liver failure and transplantation in the USA and most of Europe. About 30,000 patients per year develop acetaminophen (APAP)-induced liver injury and 29% of them undergo liver transplantation. Currently, the standard therapy for DILI is treatment with N-acetylcysteine. This treatment however only rarely leads to successful rescue of the liver, meaning that new therapies are needed.

Magnesium in its Mg²⁺ ionic form is the most abundant divalent cation in the cell and it is required as a cofactor for about 300 enzymatic reactions (Baaij et al., Physiol. Rev. 1920, 1-46 (2015)). Several proteins participate in this process allowing the flux of the cation across cell membranes (Jahnen-Dechent Clin. Kidney J. 5, i3-i14 (2012); Funato and Miki, J. Biochem. 165(3):219-225 (2019)). Magnesium homeostasis is maintained by a balance between renal reabsorption and urinary excretion, together with adequate daily intake. Magnesium dysregulation is present in several comorbidities of NAFLD such as insulin resistance and diabetes, cardiovascular complications and obesity (Özcan et al., Science (80). 306, 457 LP - 461 (2004)). Furthermore, deficiencies in Mg²⁺ are also related to triggering of the inflammatory response, mitochondrial dysfunction and decrease of antioxidant system activity (Barbagallo et al. Metabolism. 63, 502-509 (2014)). Cyclin M (CNNM) proteins play a key role in the transport of magnesium ions through cell membranes in different organs (Funato and Miki 2019). The inventors have surprisingly found that CNNM4 is overexpressed in the liver of patients with liver diseases such as NASH, cirrhosis and hepatocellular carcinoma (HCC) as well as in preclinical models for NASH, drug induced liver injury (DILI) and HCC. It may thereby cause magnesium perturbation and contribute to the cause and progression of diseases associated with a dysregulation of this cation, such as many types of liver diseases. The inventors have also found that CNNM4 inhibition in the liver reduces lipid content in preclinical models of NAFLD and can thereby be beneficial in the treatment of NAFLD. The effect is likely achieved at least in part by restoration of Mg²⁺ homeostasis in the liver. Reducing the expression of CNNM4 is therefore a promising new therapeutic strategy for treating pathologies related to the liver, and especially those associated with magnesium perturbation such as NAFLD, liver fibrosis, HCC and metabolic syndrome.

Current treatments for NAFLD focus on changing unhealthy life-style habits of patients, such as by encouraging them to take up exercise programmes and to reduce their weight. However, long-term compliance by patients is often problematic. There is therefore a need for pharmacological interventions for effective treatment of such diseases and other liver diseases.

Renal or kidney fibrosis results from an excessive accumulation of extracellular matrix (ECM), which occurs in almost every kind of chronic liver disease. The pathogenesis is a progressive process that leads to end-stage renal failure, a devastating disorder that requires dialysis or kidney transplantation. The inventors have also found CNNM4 overexpression in renal fibrosis animal models. Renal fibrosis may therefore be treated in part by inhibiting CNNM4 in the liver and/or in the kidneys.

The inventors have surprisingly also observed CNNM4 overexpression in lung cancer, and CNNM4 inhibition may be a new way of treating such a cancer.

In summary, the inventors have surprisingly found that CNNM4 is overexpressed in a group of different pathologies, both in human samples and in animal models. These pathologies comprise liver diseases such as NAFLD, cirrhosis and HCC, or acute liver pathologies such as DILI. CNNM4 overexpression has also been observed in kidney and lung diseases. Many of these diseases are highly prevalent but lack an effective treatment. The inventors propose using CNNM4 inhibition by RNA interference as a new way of treating these diseases and provide CNNM4 siRNAs for use in such treatments.

SUMMARY OF THE INVENTION

One aspect of the invention is a double-stranded nucleic acid for inhibiting expression of CNNM4, wherein the nucleic acid comprises a first strand and a second strand, wherein the first strand sequence comprises a sequence of at least 15 nucleotides differing by no more than 3 nucleotides from any one of the sequences selected from SEQ ID NO: 243, 267, 277, 279, 287, 317, 319, 325, 333, 345, 347, 349, 361, 367, 369, 371, 377, 401, 411, 413, 415, 420, 421, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 549, 550, 551 and 552.

One aspect relates to a double-stranded nucleic acid that is capable of inhibiting expression of CNNM4, preferably in a cell, for use as a medicament or in associated diagnostic or therapeutic methods, wherein the nucleic acid preferably comprises or consists of a first strand and a second strand and preferably wherein the first strand comprises a sequence sufficiently complementary to a CNNM4 mRNA so as to mediate RNA interference.

One aspect relates to a composition comprising a nucleic acid as disclosed herein and a solvent (preferably water) and/or a delivery vehicle and/or a physiologically acceptable excipient and/or a carrier and/or a salt and/or a diluent and/or a buffer and/or a preservative.

One aspect relates to a composition comprising a nucleic acid as disclosed herein and a further therapeutic agent selected from e.g., an oligonucleotide, a small molecule, a monoclonal antibody, a polyclonal antibody and a peptide.

One aspect relates to a nucleic acid or a composition comprising it as disclosed herein for use as a medicament or in associated methods.

One aspect relates to a nucleic acid or a composition comprising it as disclosed herein for use in the prevention, decrease of the risk of suffering from, or treatment of a disease, disorder or syndrome.

One aspect relates to the use of a nucleic acid or a composition comprising it as disclosed herein in the prevention, decrease of the risk of suffering from, or treatment of a disease, disorder or syndrome, wherein the disease, disorder or syndrome is preferably NASH.

One aspect relates to a method of preventing, decreasing the risk of suffering from, or treating a disease, disorder or syndrome comprising administering a pharmaceutically effective dose or amount of a nucleic acid or composition comprising it as disclosed herein to an individual in need of treatment, preferably wherein the nucleic acid or composition is administered to the subject subcutaneously, intravenously or by oral, rectal, pulmonary, intramuscular or intraperitoneal administration.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a nucleic acid which is double-stranded and which comprises a sequence homologous to and/or complementary to a portion of an expressed RNA transcript of CNNM4, and compositions thereof. These nucleic acids, or conjugates or compositions thereof, may be used in the treatment and prevention of a variety of diseases, disorders and syndromes in which reduced expression of the CNNM4 gene product is desirable.

A first aspect of the invention is a double-stranded nucleic acid for inhibiting expression of CNNM4, preferably in a cell, wherein the nucleic acid comprises a first strand and a second strand, wherein the first strand sequence comprises a sequence of at least 15 nucleotides differing by no more than 3 nucleotides from any one of the sequences selected from SEQ ID NO: 243, 267, 277, 279, 287, 317, 319, 325, 333, 345, 347, 349, 361, 367, 369, 371, 377, 401, 411, 413, 415, 420, 421, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 549, 550, 551 and 552. These nucleic acids among others have the advantage of being active in various species that are relevant for pre-clinical and clinical development and/or of having few relevant off-target effects. Having few relevant off-target effects means that a nucleic acid specifically inhibits the intended target and does not significantly inhibit other genes or inhibits only one or few other genes at a therapeutically acceptable level.

Preferably, the first strand sequence comprises a sequence of at least 16, more preferably at least 17, yet more preferably at least 18 and most preferably all 19 nucleotides differing by no more than 3 nucleotides, preferably by no more than 2 nucleotides, more preferably by no more than 1 nucleotide, and most preferably not differing by any nucleotide from any one of the sequences selected from SEQ ID NO: 243, 267, 277, 279, 287, 317, 319, 325, 333, 345, 347, 349, 361, 367, 369, 371, 377, 401, 411, 413, 415, 420, 421, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 549, 550, 551 and 552.

Preferably, the first strand sequence of the nucleic acid consists of one of the sequences selected from SEQ ID NOs: 243, 267, 277, 279, 287, 317, 319, 325, 333, 345, 347, 349, 361, 367, 369, 371, 377, 401, 411, 413, 415, 420, 421, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 549, 550, 551 and 552. The sequence may however be modified by a number of nucleic acid modifications that do not change the identity of the nucleotide. For example, modifications of the backbone or sugar residues of the nucleic acid do not change the identity of the nucleotide because the base itself remains the same as in the reference sequence.

A nucleic acid that comprises a sequence according to a reference sequence herein means that the nucleic acid comprises a sequence of contiguous nucleotides in the order as defined in the reference sequence.

When reference is made herein to a reference sequence comprising or consisting of a number of nucleotides that are not shown to be modified in that sequence, the same reference also encompasses the same nucleotide sequence in which one, several, such as two, three, four, five, six, seven or more, including all, nucleotides are modified by modifications such as 2′-OMe, 2′-F, are linked to a ligand, or a linker, have a 3′ end or 5′ end modification or any other modification. It also encompasses sequences in which two or more nucleotides are linked to each other by the natural phosphodiester linkage or by any other linkage such as a phosphorothioate or a phosphorodithioate linkage.

A double-stranded nucleic acid is a nucleic acid in which the first strand and the second strand hybridise to each other over at least part of their lengths and are therefore capable of forming a duplex region under physiological conditions, such as in PBS at 37° C. at a concentration of 1 µM of each strand. The first and second strand are preferably able to hybridise to each other and therefore to form a duplex region over a region of at least 15 nucleotides, preferably 16, 17, 18 or 19 nucleotides. This duplex region comprises nucleotide base parings between the two strands, preferably based on Watson-Crick base pairing and/or wobble base pairing (such as GU base pairing). All the nucleotides of the two strands within a duplex region do not have to base pair to each other to form a duplex region. A certain number of mismatches, deletions or insertions between the nucleotide sequences of the two strands are acceptable. Overhangs on either end of the first or second strand or unpaired nucleotides at either end of the double-stranded nucleic acid are also possible. The double-stranded nucleic acid is preferably a stable double-stranded nucleic acid under physiological conditions, and preferably has a melting temperature (Tm) of 45° C. or more, preferably 50° C. or more, and more preferably 55° C. or more for example in PBS at a concentration of 1 µM of each strand.

A stable double-stranded nucleic acid under physiological conditions is a double-stranded nucleic acid that has a Tm of 45° C. or more, preferably 50° C. or more, and more preferably 55° C. or more, for example in PBS at a concentration of 1 µM of each strand.

The first strand and the second strand are preferably capable of forming a duplex region (i.e., are complementary to each other) over i) at least a portion of their lengths, preferably over at least 15 nucleotides of both of their lengths, ii) over the entire length of the first strand, iii) over the entire length of the second strand or iv) over the entire length of both the first and the second strand. Strands being complementary to each other over a certain length means that the strands are able to base pair to each other, either via Watson-Crick or wobble base pairing, over that length. Each nucleotide of the length does not necessarily have to be able to base pair with its counterpart in the other strand over the entire given length as long as a stable double-stranded nucleotide under physiological conditions can be formed. It is however, preferred, in certain embodiments, if each nucleotide of the length can base pair with its counterpart in the other strand over the entire given length.

A certain number of mismatches, deletions or insertions between the first strand and the target sequence, or between the first strand and the second strand can be tolerated in the context of the siRNA and even have the potential in certain cases to increase RNA interference (e.g., inhibition) activity.

The inhibition activity of the nucleic acids according to the present invention relies on the formation of a duplex region between all or a portion of the first strand and a portion of a target nucleic acid. The portion of the target nucleic acid that forms a duplex region with the first strand, defined as beginning with the first base pair formed between the first strand and the target sequence and ending with the last base pair formed between the first strand and the target sequence, inclusive, is the target nucleic acid sequence or simply, target sequence. The duplex region formed between the first strand and the second strand need not be the same as the duplex region formed between the first strand and the target sequence. That is, the second strand may have a sequence different from the target sequence; however, the first strand must be able to form a duplex structure with both the second strand and the target sequence, at least under physiological conditions.

The complementarity between the first strand and the target sequence may be perfect (i.e., 100% identity with no nucleotide mismatches or insertions or deletions in the first strand as compared to the target sequence).

The complementarity between the first strand and the target sequence may not be perfect. The complementarity may be from about 70% to about 100%. More specifically, the complementarity may be at least 70%, 80%, 85%, 90% or 95% and intermediate values.

The identity between the first strand and the complementary sequence of the target sequence may range from about 75% to about 100%. More specifically, the complementarity may be at least 75%, 80%, 85%, 90% or 95% and intermediate values, provided a nucleic acid is capable of reducing or inhibiting the expression of CNNM4.

A nucleic acid having less than 100% complementarity between the first strand and the target sequence may be able to reduce the expression of CNNM4 to the same level as a nucleic acid having perfect complementarity between the first strand and target sequence. Alternatively, it may be able to reduce expression of CNNM4 to a level that is 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% of the level of reduction achieved by the nucleic acid with perfect complementarity.

In one aspect, a nucleic acid of the present disclosure is a nucleic acid wherein

-   (a) the first strand sequence comprises a sequence differing by no     more than 3 nucleotides from any one of the first strand sequences     of Table 1 and optionally wherein the second strand sequence     comprises a sequence differing by no more than 3 nucleotides from     the second strand sequence in the same line of the table; -   (b) the first strand sequence comprises a sequence differing by no     more than 2 nucleotides from any one of the first strand sequences     of Table 1 and optionally wherein the second strand sequence     comprises a sequence differing by no more than 2 nucleotides from     the second strand sequence in the same line of the table; -   (c) the first strand sequence comprises a sequence differing by no     more than 1 nucleotide from any one of the first strand sequences of     Table 1 and optionally wherein the second strand sequence comprises     a sequence differing by no more than 1 nucleotide from the second     strand sequence in the same line of the table; -   (d) the first strand sequence comprises a sequence corresponding to     nucleotides 2 to 17 from the 5′ end of any one of the first strand     sequences of Table 1 and optionally wherein the second strand     sequence comprises a sequence corresponding to nucleotides 2 to 17     from the 5′ end of the second strand sequence in the same line of     the table; -   (e) the first strand sequence comprises a sequence corresponding to     nucleotides 2 to 18 from the 5′ end of any one of the first strand     sequences of Table 1 and optionally wherein the second strand     sequence comprises a sequence corresponding to nucleotides 2 to 18     from the 5′ end of the second strand sequence in the same line of     the table; -   (f) the first strand sequence comprises a sequence corresponding to     nucleotides 2 to 19 from the 5′ end of any one of the first strand     sequences of Table 1 and optionally wherein the second strand     sequence comprises a sequence corresponding to nucleotides 2 to 19     from the 5′ end of the second strand sequence in the same line of     the table; -   (g) the first strand sequence comprises a sequence corresponding to     nucleotides 2 to 19 from the 5′ end of any one of the first strand     sequences of Table 1 and optionally wherein the second strand     sequence comprises a sequence corresponding to nucleotides 1 to 18     from the 5′ end of the second strand sequence in the same line of     the table; -   (h) the first strand sequence comprises a sequence of any one of the     first strand sequences of Table 1 and optionally wherein the second     strand sequence comprises a sequence of the second strand sequence     in the same line of the table; or -   (i) the first strand sequence consists of any one of the first     strand sequences of Table 1 and optionally wherein the second strand     sequence consists of the sequence of the second strand sequence in     the same line of the table;

wherein Table 1 is:

TABLE 1 First strand sequence (SEQ ID NO:) Second strand sequence (SEQ ID NO:) 239 240 or 374 241 242 or 376 243 244 or 378 245 246 or 379 247 248 or 380 249 250 or 386 251 252 253 254 or 388 255 256 or 390 257 258 or 392 259 260 or 394 261 262 or 396 263 264 or 398 265 266 or 400 267 268 or 402 269 270 or 404 271 272 or 406 273 274 or 408 275 276 or 410 277 278 or 412 279 280 or 414 281 282 or 416 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337 338 339 340 341 342 342 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 240 or 374 375 242 or 376 377 244 or 378 379 246 or 380 381 248 or 382 383 384 385 250 or 386 387 254 or 388 389 256 or 390 391 258 or 392 393 260 or 394 395 262 or 396 397 264 or 398 399 266 or 400 401 268 or 402 403 270 or 404 405 272 or 406 407 274 or 408 409 276 or 410 411 278 or 412 413 280 or 414 415 282 or 416 417 318 418 320 419 346 420 362 421 370 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 524 525 526 527 528 or 548 529 530 531 532 or 549 533 534 535 536 537 538 539 540 or 550 541 542 or 551 543 544 545 546 or 552 547

In one aspect, the nucleic acid is a nucleic acid wherein:

-   (a) the first strand sequence comprises the sequence of SEQ ID NO     325 and optionally wherein the second strand sequence comprises the     sequence of SEQ ID NO: 326; or -   (b) the first strand sequence comprises the sequence of SEQ ID NO     371 and optionally wherein the second strand sequence comprises the     sequence of SEQ ID NO: 372; or -   (c) the first strand sequence comprises the sequence of SEQ ID NO     411 and optionally wherein the second strand sequence comprises the     sequence of SEQ ID NO: 278 or 412; or -   (d) the first strand sequence comprises the sequence of SEQ ID NO     413 and optionally wherein the second strand sequence comprises the     sequence of SEQ ID NO: 280 or 414; or -   (e) the first strand sequence comprises the sequence of SEQ ID NO     420 and optionally wherein the second strand sequence comprises the     sequence of SEQ ID NO: 362; or -   (f) the first strand sequence comprises the sequence of SEQ ID NO     421 and optionally wherein the second strand sequence comprises the     sequence of SEQ ID NO: 370; or -   (g) the first strand sequence consists of SEQ ID NO: 371 and     optionally wherein the second strand sequence consists of SEQ ID NO:     372; or -   (h) the first strand sequence consists of SEQ ID NO: 411 and     optionally wherein the second strand sequence consists of SEQ ID NO:     278 or 412; or -   (i) the first strand sequence consists of SEQ ID NO: 413 and     optionally wherein the second strand sequence consists of SEQ ID NO:     280 or 414; or -   (j) the first strand sequence consists of SEQ ID NO: 420 and     optionally wherein the second strand sequence consists of SEQ ID NO:     362; or -   (k) the first strand sequence consists of SEQ ID NO: 421 and     optionally wherein the second strand sequence consists of SEQ ID NO:     370; or -   (l) the first strand sequence consists of SEQ ID NO: 548 and     optionally wherein the second strand sequence consists of SEQ ID NO:     529; or -   (m) the first strand sequence consists of SEQ ID NO: 549 and     optionally wherein the second strand sequence consists of SEQ ID NO:     533; or -   (n) the first strand sequence consists of SEQ ID NO: 550 and     optionally wherein the second strand sequence consists of SEQ ID NO:     541; or -   (o) the first strand sequence consists of SEQ ID NO: 551 and     optionally wherein the second strand sequence consists of SEQ ID NO:     543; or -   (p) the first strand sequence consists of SEQ ID NO: 548 and     optionally wherein the second strand sequence consists of SEQ ID NO:     547.

In one aspect, if the 5′-most nucleotide of the first strand is a nucleotide other than A or U, this nucleotide is replaced by an A or U. Preferably, if the 5′-most nucleotide of the first strand is a nucleotide other than U, this nucleotide is replaced by U, and more preferably by U with a 5′ vinylphosphonate.

In one aspect, there is a mismatch between the first nucleotide at the 5′ end of the first strand and the corresponding nucleotide (the nucleotide with which it would form a base pair if there was no mismatch) in the second strand. For example, the 5′ nucleotide of the first strand may be a U and the corresponding nucleotide in the second strand may be any nucleotide other than an A. In this case, the two nucleotides are unable to form a classical Watson-Crick base pair and there is a mismatch between the two nucleotides.

When a nucleic acid of the invention does not comprise the entire sequence of a reference first strand and/or second strand sequence, as for example given in Table 1, or one or both strands differ from the corresponding reference sequence by one, two or three nucleotides, this nucleic acid preferably retains at least 30%, more preferably at least 50%, more preferably at least 70%, more preferably at least 80%, even more preferably at least 90%, yet more preferably at least 95% and most preferably at least 100% of the CNNM4 inhibition activity compared to the inhibition activity of the corresponding nucleic acid that comprises the entire first strand and second strand reference sequences in a comparable experiment.

In one aspect, the nucleic acid is a nucleic acid wherein the first strand sequence comprises, or preferably consists of, the sequence of SEQ ID NO: 325 and optionally wherein the second strand sequence comprises, or consists of, a sequence of at least 15, preferably at least 16, more preferably at least 17, yet more preferably at least 18 and most preferably all nucleotides of the sequence of SEQ ID NO: 326; or wherein the first strand sequence comprises, or preferably consists of, the sequence of SEQ ID NO: 371 and optionally wherein the second strand sequence comprises, or consists of, a sequence of at least 15, preferably at least 16, more preferably at least 17, yet more preferably at least 18 and most preferably all nucleotides of the sequence of SEQ ID NO: 372; or wherein the first strand sequence comprises, or preferably consists of, the sequence of SEQ ID NO: 411 and optionally wherein the second strand sequence comprises, or consists of, a sequence of at least 15, preferably at least 16, more preferably at least 17, yet more preferably at least 18 and most preferably all nucleotides of the sequence of SEQ ID NO: 478 or 412; or wherein the first strand sequence comprises, or preferably consists of, the sequence of SEQ ID NO: 412 and optionally wherein the second strand sequence comprises, or consists of, a sequence of at least 15, preferably at least 16, more preferably at least 17, yet more preferably at least 18 and most preferably all nucleotides of the sequence of SEQ ID NO: 280 or 414; or wherein the first strand sequence comprises, or preferably consists of, the sequence of SEQ ID NO: 420 and optionally wherein the second strand sequence comprises, or consists of, a sequence of at least 15, preferably at least 16, more preferably at least 17, yet more preferably at least 18 and most preferably all nucleotides of the sequence of SEQ ID NO: 362; or wherein the first strand sequence comprises, or preferably consists of, the sequence of SEQ ID NO: 421 and optionally wherein the second strand sequence comprises, or consists of, a sequence of at least 15, preferably at least 16, more preferably at least 17, yet more preferably at least 18 and most preferably all nucleotides of the sequence of SEQ ID NO: 370; or wherein the first strand sequence comprises, or preferably consists of, the sequence of SEQ ID NO: 548 and optionally wherein the second strand sequence comprises, or consists of, a sequence of at least 15, preferably at least 16, more preferably at least 17, yet more preferably at least 18 and most preferably all nucleotides of the sequence of SEQ ID NO: 529; or wherein the first strand sequence comprises, or preferably consists of, the sequence of SEQ ID NO: 549 and optionally wherein the second strand sequence comprises, or consists of, a sequence of at least 15, preferably at least 16, more preferably at least 17, yet more preferably at least 18 and most preferably all nucleotides of the sequence of SEQ ID NO: 533; or wherein the first strand sequence comprises, or preferably consists of, the sequence of SEQ ID NO: 550 and optionally wherein the second strand sequence comprises, or consists of, a sequence of at least 15, preferably at least 16, more preferably at least 17, yet more preferably at least 18 and most preferably all nucleotides of the sequence of SEQ ID NO: 541; or wherein the first strand sequence comprises, or preferably consists of, the sequence of SEQ ID NO: 551 and optionally wherein the second strand sequence comprises, or consists of, a sequence of at least 15, preferably at least 16, more preferably at least 17, yet more preferably at least 18 and most preferably all nucleotides of the sequence of SEQ ID NO: 543; or wherein the first strand sequence comprises, or preferably consists of, the sequence of SEQ ID NO: 552 and optionally wherein the second strand sequence comprises, or consists of, a sequence of at least 15, preferably at least 16, more preferably at least 17, yet more preferably at least 18 and most preferably all nucleotides of the sequence of SEQ ID NO: 547.

In one aspect, the nucleic acid is a double-stranded nucleic acid for inhibiting expression of CNNM4, preferably in a cell, wherein the nucleic acid comprises a first nucleic acid strand and a second nucleic acid strand, wherein the first strand is capable of hybridising under physiological conditions to a nucleic acid of a sequence selected from SEQ ID NO: 240, 242, 244, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 302, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545 and 547; and

wherein the second strand is capable of hybridising under physiological conditions to the first strand to form a duplex region.

Nucleic acids that are capable of hybridising under physiological conditions are nucleic acids that are capable of forming base pairs, preferably Watson-Crick or wobble base-pairs, between at least a portion of the opposed nucleotides in the strands so as to form at least a duplex region. Such a double-stranded nucleic acid is preferably a stable double-stranded nucleic acid under physiological conditions (for example in PBS at 37° C. at a concentration of 1 µM of each strand), meaning that under such conditions, the two strands stay hybridised to each other. The Tm of the double-stranded nucleotide is preferably 45° C. or more, preferably 50° C. or more and more preferably 55° C. or more.

One aspect of the present invention relates to a nucleic acid for inhibiting expression of CNNM4, preferably in a cell, wherein the nucleic acid comprises a first sequence of at least 15, preferably at least 16, more preferably at least 17, yet more preferably at least 18 and most preferably all nucleotides differing by no more than 3 nucleotides, preferably no more than 2 nucleotides, more preferably no more than 1 nucleotide and most preferably not differing by any nucleotide from any of the sequences of Table 4, the first sequence being able to hybridise to a CNNM4 gene transcript (such as an mRNA) under physiological conditions. Preferably, the nucleic acid further comprises a second sequence of at least 15, preferably at least 16, more preferably at least 17, yet more preferably at least 18 and most preferably all nucleotides differing by no more than 3 nucleotides, preferably no more than 2 nucleotides, more preferably no more than 1 nucleotide and most preferably not differing by any nucleotide from any of the sequences of Table 4, wherein the second sequence is able to hybridise to the first sequence under physiological conditions and preferably wherein the nucleic acid is an siRNA that is capable of inhibiting CNNM4 expression via the RNAi pathway.

One aspect relates to any double-stranded nucleic acid as disclosed in Table 2, preferably for inhibiting expression of CNNM4, provided that the double-stranded nucleic acid is capable of inhibiting expression of CNNM4. These nucleic acids are all siRNAs with various nucleotide modifications. Some of them are conjugates comprising GalNAc moieties that can be specifically targeted to cells with GalNAc receptors, such as hepatocytes.

One aspect relates to a double-stranded nucleic acid that is capable of inhibiting expression of CNNM4, preferably in a cell, for use as a medicament or in associated diagnostic or therapeutic methods, wherein the nucleic acid preferably comprises or consists of a first strand and a second strand and preferably wherein the first strand comprises a sequence sufficiently complementary to a CNNM4 mRNA so as to mediate RNA interference.

The nucleic acids described herein may be capable of inhibiting the expression of CNNM4, preferably in a cell. The nucleic acids may be capable of inhibiting CNNM4 expression completely, resulting in 0% remaining expression upon treatment with the nucleic acids. The nucleic acids may be capable of partially inhibiting CNNM4 expression. Partial inhibition means that CNNM4 expression is decreased by 15%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95% or more, or intermediate values, as compared to the absence of a nucleic acid of the invention under comparable conditions. The level of inhibition may be measured by comparing a treated sample with an untreated sample or with a sample treated with a control such as for example a siRNA that does not target CNNM4. Inhibition may be measured by measuring CNNM4 mRNA and/or protein levels or levels of a biomarker or indicator that correlates with CNNM4 presence or activity. It may be measured in cells that may have been treated in vitro with a nucleic acid described herein. Alternatively, or in addition, inhibition may be measured in cells, such as hepatocytes, or tissue, such as liver tissue, or an organ, such as the liver, or in a body fluid such as blood, serum, lymph or in any other body part or fluid that has been taken from a subject previously treated with a nucleic acid disclosed herein. Preferably, inhibition of CNNM4 expression is determined by comparing the CNNM4 mRNA level measured in CNNM4-expressing cells after 24 or 48 hours in vitro treatment with a double-stranded RNA disclosed herein under ideal conditions (see the examples for appropriate concentrations and conditions) to the CNNM4 mRNA level measured in control cells that were untreated or mock treated or treated with a control double-stranded RNA under the same or at least comparable conditions.

One aspect of the present invention relates to a nucleic acid, wherein the first strand and the second strand are present on a single strand of a nucleic acid that loops around so that the first strand and the second strand are able to hybridise to each other and to thereby form a double-stranded nucleic acid with a duplex region.

Preferably, the first strand and the second strand of the nucleic acid are separate strands. The two separate strands are preferably each 17-25 nucleotides in length, more preferably 18-25 nucleotides in length. The two strands may be of the same or different lengths. The first strand may be 17-25 nucleotides in length, preferably it may be 18-24 nucleotides in length, it may be 18, 19, 20, 21, 22, 23 or 24 nucleotides in length. Most preferably, the first strand is 19 nucleotides in length. The second strand may independently be 17-25 nucleotides in length, preferably it may be 18-24 nucleotides in length, it may be 18, 19, 20, 21, 22, 23 or 24 nucleotides in length. More preferably, the second strand is 18 or 19 or 20 nucleotides in length, and most preferably it is 19 nucleotides in length.

Preferably, the first strand and the second strand of the nucleic acid form a duplex region of 17-25 nucleotides in length. More preferably, the duplex region is 18-24 nucleotides in length. The duplex region may be 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length. In the most preferred embodiment, the duplex region is 18 or 19 nucleotides in length. The duplex region is defined here as the region between and including the 5′-most nucleotide of the first strand that is base paired to a nucleotide of the second strand to the 3′-most nucleotide of the first strand that is base paired to a nucleotide of the second strand. The duplex region may comprise nucleotides in either or both strands that are not base-paired to a nucleotide in the other strand. It may comprise one, two, three or four such nucleotides on the first strand and/or on the second strand. However, preferably, the duplex region consists of 17-25 consecutive nucleotide base pairs. That is to say that it preferably comprises 17-25 consecutive nucleotides on both of the strands that all base pair to a nucleotide in the other strand. More preferably, the duplex region consists of 18 or 19 consecutive nucleotide base pairs, most preferably 18.

In each of the embodiments disclosed herein, the nucleic acid may be blunt ended at both ends; have an overhang at one end and a blunt end at the other end; or have an overhang at both ends.

The nucleic acid may have an overhang at one end and a blunt end at the other end. The nucleic acid may have an overhang at both ends. The nucleic acid may be blunt ended at both ends. The nucleic acid may be blunt ended at the end with the 5′ end of the first strand and the 3′ end of the second strand or at the 3′ end of the first strand and the 5′ end of the second strand.

The nucleic acid may comprise an overhang at a 3′ or 5′ end. The nucleic acid may have a 3′ overhang on the first strand. The nucleic acid may have a 3′ overhang on the second strand. The nucleic acid may have a 5′ overhang on the first strand. The nucleic acid may have a 5′ overhang on the second strand. The nucleic acid may have an overhang at both the 5′ end and 3′ end of the first strand. The nucleic acid may have an overhang at both the 5′ end and 3′ end of the second strand. The nucleic acid may have a 5′ overhang on the first strand and a 3′ overhang on the second strand. The nucleic acid may have a 3′ overhang on the first strand and a 5′ overhang on the second strand. The nucleic acid may have a 3′ overhang on the first strand and a 3′ overhang on the second strand. The nucleic acid may have a 5′ overhang on the first strand and a 5′ overhang on the second strand.

An overhang at the 3′ end or 5′ end of the second strand or the first strand may consist of 1, 2, 3, 4 and 5 nucleotides in length. Optionally, an overhang may consist of 1 or 2 nucleotides, which may or may not be modified.

In one embodiment, the 5′ end of the first strand is a single-stranded overhang of one, two or three nucleotides, preferably of one nucleotide.

Preferably, the nucleic acid is an siRNA. siRNAs are short interfering or short silencing RNAs that are able to inhibit the expression of a target gene through the RNA interference (RNAi) pathway. Inhibition occurs through targeted degradation of mRNA transcripts of the target gene after transcription. The siRNA forms part of the RISC complex. The RISC complex specifically targets the target RNA by sequence complementarity of the first (antisense) strand with the target sequence.

Preferably, the nucleic acid is capable of inhibiting CNNM4. The inhibition is preferably mediated by the RNA interference (RNAi) mechanism. Preferably, the nucleic acid mediates RNA interference (i.e., it is capable of inhibiting its target) with an efficacy of at least 50% inhibition, more preferably at least 70%, more preferably at least 80%, even more preferably at least 90%, yet more preferably at least 95% and most preferably 100% inhibition. The inhibition efficacy is preferably measured by comparing the CNNM4 mRNA level in cells, such as hepatocytes, treated with a CNNM4-specific siRNA to the CNNM4 mRNA level in cells treated with a control in a comparable experiment. The control can be a treatment with a non-CNNM4 targeting siRNA or without a siRNA. The nucleic acid, or at least the first strand of the nucleic acid, is therefore preferably able to be incorporated into the RISC complex. As a result, the nucleic acid, or at least the first strand of the nucleic acid, is therefore able to guide the RISC complex to a specific target RNA with which the nucleic acid, or at least the first strand of the nucleic acid, is at least partially complementary. The RISC complex then specifically cleaves this target RNA and as a result leads to inhibition of the expression of the gene from which the RNA stems.

Nucleic Acid Modifications

Nucleic acids discussed herein include unmodified RNA as well as RNA which has been modified, e.g., to improve efficacy or stability. Unmodified RNA refers to a molecule in which the components of the nucleic acid, namely sugars, bases, and phosphate moieties, are the same or essentially the same as those which occur in nature, for example as occur naturally in the human body. The term “modified nucleotide” as used herein refers to a nucleotide in which one or more of the components of the nucleotide, namely the sugar, base, and phosphate moiety, is/are different from those which occur in nature. The term “modified nucleotide” also refers in certain cases to molecules that are not nucleotides in the strict sense of the term because they lack, or have a substitute of, an essential component of a nucleotide, such as the sugar, base or phosphate moiety. A nucleic acid comprising such modified nucleotides is still to be understood as being a nucleic acid, even if one or more of the nucleotides of the nucleic acid has been replaced by a modified nucleotide that lacks, or has a substitution of, an essential component of a nucleotide.

Modifications of the nucleic acid of the present invention generally provide a powerful tool in overcoming potential limitations including, but not limited to, in vitro and in vivo stability and bioavailability inherent to native RNA molecules. The nucleic acids according to the invention may be modified by chemical modifications. Modified nucleic acids can also minimise the possibility of inducing interferon activity in humans. Modifications can further enhance the functional delivery of a nucleic acid to a target cell. The modified nucleic acids of the present invention may comprise one or more chemically modified ribonucleotides of either or both of the first strand or the second strand. A ribonucleotide may comprise a chemical modification of the base, sugar or phosphate moieties. The ribonucleic acid may be modified by substitution with or insertion of analogues of nucleic acids or bases.

Throughout the description of the invention, “same or common modification” means the same modification to any nucleotide, be that A, G, C or U modified with a group such as a methyl group (2′-OMe) or a fluoro group (2′-F). For example, 2′-F-dU, 2′-F-dA, 2′-F-dC, 2′-F-dG are all considered to be the same or common modification, as are 2′-OMe-rU, 2′-OMe-rA; 2′-OMe-rC; 2′-OMe-rG. In contrast, a 2′-F modification is a different modification compared to a 2′-OMe modification.

Preferably, at least one nucleotide of the first and/or second strand of the nucleic acid is a modified nucleotide, preferably a non-naturally occurring nucleotide such as preferably a 2′-F modified nucleotide.

A modified nucleotide can be a nucleotide with a modification of the sugar group. The 2′ hydroxyl group (OH) can be modified or replaced with a number of different “oxy” or “deoxy” substituents.

Examples of “oxy″-2′ hydroxyl group modifications include alkoxy or aryloxy (OR, e.g., R=H, alkyl (such as methyl), cycloalkyl, aryl, aralkyl, heteroaryl or sugar); polyethyleneglycols (PEG), O(CH₂CH₂O)_(n)CH₂CH₂OR; “locked” nucleic acids (LNA) in which the 2′ hydroxyl is connected, e.g., by a methylene bridge, to the 4′ carbon of the same ribose sugar; O-AMINE (AMINE=NH₂, alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino, ethylene diamine, or polyamino) and aminoalkoxy, O(CH₂)nAMINE, (e.g., AMINE=NH₂, alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino, ethylene diamine, or polyamino).

“Deoxy” modifications include hydrogen, halogen, amino (e.g., NH₂, alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, or amino acid); NH(CH₂CH₂NH)_(n)CH₂CH₂-AMINE (AMINE=NH₂, alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino), —NHC(O)R (R=alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), cyano; mercapto; alkyl-thio-alkyl; thioalkoxy; and alkyl, cycloalkyl, aryl, alkenyl and alkynyl, which may be optionally substituted with e.g., an amino functionality. Other substituents of certain embodiments include 2′-methoxyethyl, 2′-OCH₃, 2′-O-allyl, 2′-C-allyl, and 2′-fluoro.

The sugar group can also contain one or more carbons that possess the opposite stereochemical configuration than that of the corresponding carbon in ribose. Thus, a modified nucleotide may contain a sugar such as arabinose.

Modified nucleotides can also include “abasic” sugars, which lack a nucleobase at C - 1′. These abasic sugars can further contain modifications at one or more of the constituent sugar atoms.

The 2′ modifications may be used in combination with one or more phosphate internucleoside linker modifications (e.g., phosphorothioate or phosphorodithioate).

One or more nucleotides of a nucleic acid of the present invention may be modified. The nucleic acid may comprise at least one modified nucleotide. The modified nucleotide may be in the first strand. The modified nucleotide may be in the second strand. The modified nucleotide may be in the duplex region. The modified nucleotide may be outside the duplex region, i.e., in a single-stranded region. The modified nucleotide may be on the first strand and may be outside the duplex region. The modified nucleotide may be on the second strand and may be outside the duplex region. The 3′-terminal nucleotide of the first strand may be a modified nucleotide. The 3′-terminal nucleotide of the second strand may be a modified nucleotide. The 5′-terminal nucleotide of the first strand may be a modified nucleotide. The 5′-terminal nucleotide of the second strand may be a modified nucleotide.

A nucleic acid of the invention may have 1 modified nucleotide or a nucleic acid of the invention may have about 2-4 modified nucleotides, or a nucleic acid may have about 4-6 modified nucleotides, about 6-8 modified nucleotides, about 8-10 modified nucleotides, about 10-12 modified nucleotides, about 12-14 modified nucleotides, about 14-16 modified nucleotides about 16-18 modified nucleotides, about 18-20 modified nucleotides, about 20-22 modified nucleotides, about 22-24 modified nucleotides, about 24-26 modified nucleotides or about 26-28 modified nucleotides. In each case the nucleic acid comprising said modified nucleotides retains at least 50% of its activity as compared to the same nucleic acid but without said modified nucleotides or vice versa. The nucleic acid may retain 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% and intermediate values of its activity as compared to the same nucleic acid but without said modified nucleotides, or may have more than 100% of the activity of the same nucleic acid without said modified nucleotides.

The modified nucleotide may be a purine or a pyrimidine. At least half of the purines may be modified. At least half of the pyrimidines may be modified. All of the purines may be modified. All of the pyrimidines may be modified. The modified nucleotides may be selected from the group consisting of a 3′ terminal deoxy thymine (dT) nucleotide, a 2′-O-methyl (2′-OMe) modified nucleotide, a 2′ modified nucleotide, a 2′ deoxy modified nucleotide, a locked nucleotide, an abasic nucleotide, a 2′ amino modified nucleotide, a 2′ alkyl modified nucleotide, a 2′-deoxy-2′-fluoro (2′-F) modified nucleotide, a morpholino nucleotide, a phosphoramidate, a non-natural base comprising nucleotide, a nucleotide comprising a 5′-phosphorothioate group, a nucleotide comprising a 5′ phosphate or 5′ phosphate mimic and a terminal nucleotide linked to a cholesteryl derivative or a dodecanoic acid bisdecylamide group.

The nucleic acid may comprise a nucleotide comprising a modified base, wherein the base is selected from 2-aminoadenosine, 2,6-diaminopurine,inosine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2, 4, 6-trimethoxy benzene, 3-methyl uracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidine (e.g., 5-methylcytidine), 5-alkyluridine (e.g., ribothymidine), 5-halouridine (e.g., 5-bromouridine), 6-azapyrimidine, 6-alkylpyrimidine (e.g. 6-methyluridine), propyne, quesosine, 2-thiouridine, 4-thiouridine, wybutosine, wybutoxosine, 4-acetylcytidine, 5-(carboxyhydroxymethyl)uridine, 5′-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluridine, beta-D-galactosylqueosine, 1-methyladenosine, 1-methylinosine, 2,2-dimethylguanosine, 3-methylcytidine, 2-methyladenosine, 2-methylguanosine, N6-methyladenosine, 7-methylguanosine, 5-methoxyaminomethyl-2-thiouridine, 5-methylaminomethyluridine, 5-methylcarbonylmethyluridine, 5-methyloxyuridine, 5-methyl-2-thiouridine, 2-methylthio-N6-isopentenyladenosine, beta-D-mannosylqueosine, uridine-5-oxyacetic acid and 2-thiocytidine.

Many of the modifications described herein and that occur within a nucleic acid will be repeated within a polynucleotide molecule, such as a modification of a base, or a phosphate moiety, or a non-linking O of a phosphate moiety. In some cases, the modification will occur at all of the possible positions/nucleotides in the polynucleotide but in many cases it will not. A modification may only occur at a 3′ or 5′ terminal position, may only occur in a terminal region, such as at a position on a terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of a strand. A modification may occur in a double-strand region, a single-strand region, or in both. A modification may occur only in the double-strand region of a nucleic acid of the invention or may only occur in a single-strand region of a nucleic acid of the invention. A phosphorothioate or phosphorodithioate modification at a non-linking O position may only occur at one or both termini, may only occur in a terminal region, e.g., at a position on a terminal nucleotide or in the last 2, 3, 4 or 5 nucleotides of a strand, or may occur in duplex and/or in single-strand regions, particularly at termini. The 5′ end and/or 3′ end may be phosphorylated.

Stability of a nucleic acid of the invention may be increased by including particular bases in overhangs, or by including modified nucleotides, in single-strand overhangs, e.g., in a 5′ or 3′ overhang, or in both. Purine nucleotides may be included in overhangs. All or some of the bases in a 3′ or 5′ overhang may be modified. Modifications can include the use of modifications at the 2′ OH group of the ribose sugar, the use of deoxyribonucleotides, instead of ribonucleotides, and modifications in the phosphate group, such as phosphorothioate or phosphorodithioate modifications. Overhangs need not be homologous with the target sequence.

Nucleases can hydrolyse nucleic acid phosphodiester bonds. However, chemical modifications to nucleic acids can confer improved properties, and, can render oligoribonucleotides more stable to nucleases.

Modified nucleic acids, as used herein, can include one or more of:

-   (i) alteration, e.g., replacement, of one or both of the non-linking     phosphate oxygens and/or of one or more of the linking phosphate     oxygens (referred to as linking even if at the 5′ and 3′ terminus of     the nucleic acid of the invention); -   (ii) alteration, e.g., replacement, of a constituent of the ribose     sugar, e.g., of the 2′ hydroxyl on the ribose sugar; -   (iii) replacement of the phosphate moiety with “dephospho” linkers; -   (iv) modification or replacement of a naturally occurring base; -   (v) replacement or modification of the ribose-phosphate backbone;     and -   (vi) modification of the 3′ end or 5′ end of the first strand and/or     the second strand, e.g., removal, modification or replacement of a     terminal phosphate group or conjugation of a moiety, e.g., a     fluorescently labelled moiety, to either the 3′ or 5′ end of one or     both strands.

The terms replacement, modification and alteration indicate a difference from a naturally occurring molecule.

Specific modifications are discussed in more detail below.

The nucleic acid may comprise one or more nucleotides on the second and/or first strands that are modified. Alternating nucleotides may be modified, to form modified nucleotides.

Alternating as described herein means to occur one after another in a regular way. In other words, alternating means to occur in turn repeatedly. For example, if one nucleotide is modified, the next contiguous nucleotide is not modified and the following contiguous nucleotide is modified and so on. One nucleotide may be modified with a first modification, the next contiguous nucleotide may be modified with a second modification and the following contiguous nucleotide is modified with the first modification and so on, where the first and second modifications are different.

Some representative modified nucleic acid sequences of the present invention are shown in the examples. These examples are meant to be representative and not limiting.

In one aspect of the nucleic acid, at least nucleotides 2 and 14 of the first strand are modified, preferably by a first common modification, the nucleotides being numbered consecutively starting with nucleotide number 1 at the 5′ end of the first strand. The first modification is preferably 2′-F.

In one aspect, at least one, several or preferably all the even-numbered nucleotides of the first strand are modified, preferably by a first common modification, the nucleotides being numbered consecutively starting with nucleotide number 1 at the 5′ end of the first strand. The first modification is preferably 2′-F.

In one aspect, at least one, several or preferably all the odd-numbered nucleotides of the first strand are modified, the nucleotides being numbered consecutively starting with nucleotide number 1 at the 5′ end of the first strand. Preferably, they are modified by a second modification. This second modification is preferably different from the first modification if the nucleic acid also comprises a first modification, for example of nucleotides 2 and 14 or of all the even-numbered nucleotides of the first strand. The first modification is preferably any 2′ ribose modification that is of the same size or smaller in volume than a 2′-OH group, or a locked nucleic acid (LNA), or an unlocked nucleic acid (UNA), or a 2′-Fluoroarabino Nucleic Acid (FANA) modification. A 2′ ribose modification that is of the same size or smaller in volume than a 2′-OH group can for example be a 2′-F, 2′-H, 2′-halo, or 2′-NH₂. The second modification is preferably any 2′ ribose modification that is larger in volume than a 2′-OH group. A 2′ ribose modification that is larger in volume than a 2′-OH group can for example be a 2′-OMe, 2′-O-MOE (2′-O-methoxyethyl), 2′-O-allyl or 2′-O-alkyl, with the proviso that the nucleic is capable of reducing the expression of the target gene to at least the same extent as the same nucleic acid without the modification(s) under comparable conditions. The first modification is preferably 2′-F and/or the second modification is preferably 2′-OMe.

In the context of this disclosure, the size or volume of a substituent, such as a 2′ ribose modification, is preferably measured as the van der Waals volume.

In one aspect, at least one, several or preferably all the nucleotides of the second strand in a position corresponding to an even-numbered nucleotide of the first strand are modified, preferably by a third modification. Preferably in the same nucleic acid nucleotides 2 and 14 or all the even numbered nucleotides of the first strand are modified with a first modification. In addition, or alternatively, the odd-numbered nucleotides of the first strand are modified with a second modification. Preferably, the third modification is different from the first modification and/or the third modification is the same as the second modification. The first modification is preferably any 2′ ribose modification that is of the same size or smaller in volume than a 2′-OH group, or a locked nucleic acid (LNA), or an unlocked nucleic acid (UNA), or a 2′-Fluoroarabino Nucleic Acid (FANA) modification. A 2′ ribose modification that is of the same size or smaller in volume than a 2′-OH group can for example be a 2′-F, 2′-H, 2′-halo, or 2′-NH₂. The second and/or third modification is preferably any 2′ ribose modification that is larger in volume than a 2′-OH group. A 2′ ribose modification that is larger in volume than a 2′-OH group can for example be a 2′-OMe, 2′-O-MOE (2′-O-methoxyethyl), 2′-O-allyl or 2′-O-alkyl, with the proviso that the nucleic is capable of reducing the expression of the target gene to at least the same extent as the same nucleic acid without the modification(s) under comparable conditions. The first modification is preferably 2′-F and/or the second and/or third modification is/are preferably 2′-OMe. The nucleotides on the first strand are numbered consecutively starting with nucleotide number 1 at the 5′ end of the first strand.

A nucleotide of the second strand that is in a position corresponding, for example, to an even-numbered nucleotide of the first strand is a nucleotide of the second strand that is base-paired to an even-numbered nucleotide of the first strand.

In one aspect, at least one, several or preferably all the nucleotides of the second strand in a position corresponding to an odd-numbered nucleotide of the first strand are modified, preferably by a fourth modification. Preferably in the same nucleic acid nucleotides 2 and 14 or all the even numbered nucleotides of the first strand are modified with a first modification. In addition, or alternatively, the odd-numbered nucleotides of the first strand are modified with a second modification. In addition, or alternatively, all the nucleotides of the second strand in a position corresponding to an even-numbered nucleotide of the first strand are modified with a third modification. The fourth modification is preferably different from the second modification and preferably different from the third modification and the fourth modification is preferably the same as the first modification. The first and/or fourth modification is preferably any 2′ ribose modification that is of the same size or smaller in volume than a 2′-OH group, or a locked nucleic acid (LNA), or an unlocked nucleic acid (UNA), or a 2′-Fluoroarabino Nucleic Acid (FANA) modification. A 2′ ribose modification that is of the same size or smaller in volume than a 2′-OH group can for example be a 2′-F, 2′-H, 2′-halo, or 2′-NH₂. The second and/or third modification is preferably any 2′ ribose modification that is larger in volume than a 2′-OH group. A 2′ ribose modification that is larger in volume than a 2′-OH group can for example be a 2′-OMe, 2′-O-MOE (2′-O-methoxyethyl), 2′-O-allyl or 2′-O-alkyl, with the proviso that the nucleic is capable of reducing the expression of the target gene to at least the same extent as the same nucleic acid without the modification(s) under comparable conditions. The first and/or the fourth modification is/are preferably a 2′-OMe modification and/or the second and/or third modification is/are preferably a 2′-F modification. The nucleotides on the first strand are numbered consecutively starting with nucleotide number 1 at the 5′ end of the first strand.

In one aspect of the nucleic acid, the nucleotide/nucleotides of the second strand in a position corresponding to nucleotide 11 or nucleotide 13 or nucleotides 11 and 13 or nucleotides 11-13 of the first strand is/are modified by a fourth modification. Preferably, all the nucleotides of the second strand other than the nucleotide/nucleotides in a position corresponding to nucleotide 11 or nucleotide 13 or nucleotides 11 and 13 or nucleotides 11-13 of the first strand is/are modified by a third modification. Preferably in the same nucleic acid nucleotides 2 and 14 or all the even numbered nucleotides of the first strand are modified with a first modification. In addition, or alternatively, the odd-numbered nucleotides of the first strand are modified with a second modification. The fourth modification is preferably different from the second modification and preferably different from the third modification and the fourth modification is preferably the same as the first modification. The first and/or fourth modification is preferably any 2′ ribose modification that is of the same size or smaller in volume than a 2′-OH group, or a locked nucleic acid (LNA), or an unlocked nucleic acid (UNA), or a 2′-Fluoroarabino Nucleic Acid (FANA) modification. A 2′ ribose modification that is of the same size or smaller in volume than a 2′-OH group can for example be a 2′-F, 2′-H, 2′-halo, or 2′-NH₂. The second and/or third modification is preferably any 2′ ribose modification that is larger in volume than a 2′-OH group. A 2′ ribose modification that is larger in volume than a 2′-OH group can for example be a 2′-OMe, 2′-O-MOE (2′c-O-methoxyethyl), 2′-O-allyl or 2′-O-alkyl, with the proviso that the nucleic is capable of reducing the expression of the target gene to at least the same extent as the same nucleic acid without the modification(s) under comparable conditions. The first and/or the fourth modification is/are preferably a 2′-OMe modification and/or the second and/or third modification is/are preferably a 2′-F modification. The nucleotides on the first strand are numbered consecutively starting with nucleotide number 1 at the 5′ end of the first strand.

In one aspect of the nucleic acid, all the even-numbered nucleotides of the first strand are modified by a first modification, all the odd-numbered nucleotides of the first strand are modified by a second modification, all the nucleotides of the second strand in a position corresponding to an even-numbered nucleotide of the first strand are modified by a third modification, all the nucleotides of the second strand in a position corresponding to an odd-numbered nucleotide of the first strand are modified by a fourth modification, wherein the first and/or fourth modification is/are 2′-F and/or the second and/or third modification is/are 2′-OMe.

In one aspect of the nucleic acid, all the even-numbered nucleotides of the first strand are modified by a first modification, all the odd-numbered nucleotides of the first strand are modified by a second modification, all the nucleotides of the second strand in positions corresponding to nucleotides 11-13 of the first strand are modified by a fourth modification, all the nucleotides of the second strand other than the nucleotides corresponding to nucleotides 11-13 of the first strand are modified by a third modification, wherein the first and fourth modification are 2′-F and the second and third modification are 2′-OMe. In one embodiment in this aspect, the 3′ terminal nucleotide of the second strand is an inverted RNA nucleotide (i.e., the nucleotide is linked to the 3′ end of the strand through its 3′ carbon, rather than through its 5′ carbon as would normally be the case). When the 3′ terminal nucleotide of the second strand is an inverted RNA nucleotide, the inverted RNA nucleotide is preferably an unmodified nucleotide in the sense that it does not comprise any modifications compared to the natural nucleotide counterpart. Specifically, the inverted RNA nucleotide is preferably a 2′-OH nucleotide. Preferably, in this aspect when the 3′ terminal nucleotide of the second strand is an inverted RNA nucleotide, the nucleic acid is blunt-ended at least at the end that comprises the 5′ end of the first strand.

One aspect of the present invention is a nucleic acid as disclosed herein for inhibiting expression of the CNNM4 gene, preferably in a cell, wherein said first strand includes modified nucleotides or unmodified nucleotides at a plurality of positions in order to facilitate processing of the nucleic acid by RISC.

In one aspect, “facilitate processing by RISC” means that the nucleic acid can be processed by RISC, for example any modification present will permit the nucleic acid to be processed by RISC and preferably, will be beneficial to processing by RISC, suitably such that siRNA activity can take place.

One aspect is a nucleic acid as disclosed herein, wherein the nucleotides at positions 2 and 14 from the 5′ end of the first strand are not modified with a 2′-OMe modification, and the nucleotide/nucleotides on the second strand which corresponds to position 11 or position 13 or positions 11 and 13 or positions 11, 12 and 13 of the first strand is/are not modified with a 2′-OMe modification (in other words, they are naturally occurring nucleotides or are modified with a modification other than 2′-OMe).

In one aspect, the nucleotide on the second strand which corresponds to position 13 of the first strand is the nucleotide that forms a base pair with position 13 (from the 5′ end) of the first strand.

In one aspect, the nucleotide on the second strand which corresponds to position 11 of the first strand is the nucleotide that forms a base pair with position 11 (from the 5′ end) of the first strand.

In one aspect, the nucleotide on the second strand which corresponds to position 12 of the first strand is the nucleotide that forms a base pair with position 12 (from the 5′ end) of the first strand.

For example, in a 19-mer nucleic acid which is double-stranded and blunt ended, position 13 (from the 5′ end) of the first strand would pair with position 7 (from the 5′ end) of the second strand. Position 11 (from the 5′ end) of the first strand would pair with position 9 (from the 5′ end) of the second strand. This nomenclature may be applied to other positions of the second strand.

In one aspect, in the case of a partially complementary first and second strand, the nucleotide on the second strand that “corresponds to” a position on the first strand may not necessarily form a base pair if that position is the position in which there is a mismatch, but the principle of the nomenclature still applies.

One aspect is a nucleic acid as disclosed herein, wherein the nucleotides at positions 2 and 14 from the 5′ end of the first strand are not modified with a 2′-OMe modification, and the nucleotides on the second strand which correspond to position 11, or 13, or 11 and 13, or 11-13 of the first strand are modified with a 2′-F modification.

One aspect is a nucleic acid as disclosed herein, wherein the nucleotides at positions 2 and 14 from the 5′ end of the first strand are modified with a 2′-F modification, and the nucleotides on the second strand which correspond to position 11, or 13, or 11 and 13, or 11-13 of the first strand are not modified with a 2′-OMe modification.

One aspect is a nucleic acid as disclosed herein, wherein the nucleotides at positions 2 and 14 from the 5′ end of the first strand are modified with a 2′-F modification, and the nucleotides on the second strand which correspond to position 11, or 13, or 11 and 13, or 11-13 of the first strand are modified with a 2′-F modification.

One aspect is a nucleic acid as disclosed herein wherein greater than 50% of the nucleotides of the first and/or second strand comprise a 2′-OMe modification, such as greater than 55%, 60%, 65%, 70%, 75%, 80%, or 85%, or more, of the first and/or second strand comprise a 2′-OMe modification.

One aspect is a nucleic acid as disclosed herein wherein greater than 50% of the nucleotides of the first and/or second strand comprise a naturally occurring RNA modification, such as wherein greater than 55%, 60%, 65%, 70%, 75%, 80%, or 85% or more of the first and/or second strands comprise such a modification. Suitable naturally occurring modifications include, as well as 2′-OMe, other 2′ sugar modifications, in particular a 2′-H modification resulting in a DNA nucleotide.

One aspect is a nucleic acid as disclosed herein comprising no more than 20%, such as no more than 15% such as no more than 10%, of nucleotides which have 2′ modifications that are not 2′-OMe modifications on the first and/or second strand.

One aspect is a nucleic acid as disclosed herein, wherein the number of nucleotides in the first and/or second strand with a 2′-modification that is not a 2′-OMe modification is no more than 7, more preferably no more than 5, and most preferably no more than 3.

One aspect is a nucleic acid as disclosed herein comprising no more than 20%, (such as no more than 15% or no more than 10%) of 2′-F modifications on the first and/or second strand.

One aspect is a nucleic acid as disclosed herein, wherein the number of nucleotides in the first and/or second strand with a 2′-F modification is no more than 7, more preferably no more than 5, and most preferably no more than 3.

One aspect is a nucleic acid as disclosed herein, wherein all nucleotides are modified with a 2′-OMe modification except positions 2 and 14 from the 5′ end of the first strand and the nucleotides on the second strand which correspond to position 11, or 13, or 11 and 13, or 11-13 of the first strand. Preferably the nucleotides that are not modified with 2′-OMe are modified with fluoro at the 2′ position (2′-F modification).

Preferred is a nucleic acid as disclosed herein wherein all nucleotides of the nucleic acid are modified at the 2′ position of the sugar. Preferably these nucleotides are modified with a 2′-F modification where the modification is not a 2′-OMe modification.

In one aspect the nucleic acid is modified on the first strand with alternating 2′-OMe modifications and 2-F modifications, and positions 2 and 14 (starting from the 5′ end) are modified with 2′-F. Preferably the second strand is modified with 2′-F modifications at nucleotides on the second strand which correspond to position 11, or 13, or 11 and 13, or 11-13 of the first strand. Preferably the second strand is modified with 2′-F modifications at positions 11-13 counting from the 3′ end starting at the first position of the complementary (double-stranded) region, and the remaining modifications are naturally occurring modifications, preferably 2′-OMe. The complementary region at least in this case starts at the first position of the second strand that has a corresponding nucleotide in the first strand, regardless of whether the two nucleotides are able to base pair to each other.

In one aspect of the nucleic acid, each of the nucleotides of the first strand and of the second strand is a modified nucleotide.

Unless specifically stated otherwise, herein the nucleotides of the first strand are numbered contiguously starting with nucleotide number 1 at the 5′ end of the first strand. Nucleotides of the second strand are numbered contiguously starting with nucleotide number 1 at the 3′ end of the second strand.

An “odd numbered” nucleotide is a nucleotide numbered with an odd number in a strand in which the nucleotides are numbered contiguously starting either from the indicated end or from the 5′ end of the strand if the end from which the nucleotides are numbered is not indicated. An “even numbered” nucleotide is a nucleotide numbered with an even number in a strand in which the nucleotides are numbered contiguously starting either from the indicated end or from the 5′ end of the strand if the end from which the nucleotides are numbered is not indicated.

One or more nucleotides on the first and/or second strand may be modified, to form modified nucleotides. One or more of the odd-numbered nucleotides of the first strand may be modified. One or more of the even-numbered nucleotides of the first strand may be modified by at least a second modification, wherein the at least second modification is different from the modification on the one or more odd nucleotides. At least one of the one or more modified even numbered-nucleotides may be adjacent to at least one of the one or more modified odd-numbered nucleotides.

A plurality of odd-numbered nucleotides in the first strand may be modified in the nucleic acid of the invention. A plurality of even-numbered nucleotides in the first strand may be modified by a second modification. The first strand may comprise adjacent nucleotides that are modified by a common modification. The first strand may also comprise adjacent nucleotides that are modified by a second different modification (i.e., the first strand may comprise nucleotides that are adjacent to each other and modified by a first modification as well as other nucleotides that are adjacent to each other and modified by a second modification that is different to the first modification).

One or more of the odd-numbered nucleotides of the second strand (wherein the nucleotides are numbered contiguously starting with nucleotide number 1 at the 3′ end of the second strand) may be modified by a modification that is different to the modification of the odd-numbered nucleotides on the first strand (wherein the nucleotides are numbered contiguously starting with nucleotide number 1 at the 5′ end of the first strand) and/or one or more of the even-numbered nucleotides of the second strand may be modified by the same modification of the odd-numbered nucleotides of the first strand. At least one of the one or more modified even-numbered nucleotides of the second strand may be adjacent to the one or more modified odd-numbered nucleotides. A plurality of odd-numbered nucleotides of the second strand may be modified by a common modification and/or a plurality of even-numbered nucleotides may be modified by the same modification that is present on the first stand odd-numbered nucleotides. A plurality of odd-numbered nucleotides on the second strand may be modified by a modification that is different from the modification of the first strand odd-numbered nucleotides.

The second strand may comprise adjacent nucleotides that are modified by a common modification, which may be a modification that is different from the modification of the odd-numbered nucleotides of the first strand.

In the nucleic acid of the invention, each of the odd-numbered nucleotides in the first strand and each of the even-numbered nucleotides in the second strand may be modified with a common modification and, each of the even-numbered nucleotides may be modified in the first strand with a different modification and each of the odd-numbered nucleotides may be modified in the second strand with the different modification.

The nucleic acid of the invention may have the modified nucleotides of the first strand shifted by at least one nucleotide relative to the unmodified or differently modified nucleotides of the second strand.

One or more or each of the odd numbered-nucleotides may be modified in the first strand and one or more or each of the even-numbered nucleotides may be modified in the second strand. One or more or each of the alternating nucleotides on either or both strands may be modified by a second modification. One or more or each of the even-numbered nucleotides may be modified in the first strand and one or more or each of the even-numbered nucleotides may be modified in the second strand. One or more or each of the alternating nucleotides on either or both strands may be modified by a second modification. One or more or each of the odd-numbered nucleotides may be modified in the first strand and one or more of the odd-numbered nucleotides may be modified in the second strand by a common modification. One or more or each of the alternating nucleotides on either or both strands may be modified by a second modification. One or more or each of the even-numbered nucleotides may be modified in the first strand and one or more or each of the odd-numbered nucleotides may be modified in the second strand by a common modification. One or more or each of the alternating nucleotides on either or both strands may be modified by a second modification.

The nucleic acid of the invention may comprise single- or double-stranded constructs that comprise at least two regions of alternating modifications in one or both of the strands. These alternating regions can comprise up to about 12 nucleotides but preferably comprise from about 3 to about 10 nucleotides. The regions of alternating nucleotides may be located at the termini of one or both strands of the nucleic acid of the invention. The nucleic acid may comprise from 4 to about 10 nucleotides of alternating nucleotides at each of the termini (3′ and 5′) and these regions may be separated by from about 5 to about 12 contiguous unmodified or differently or commonly modified nucleotides.

The odd numbered nucleotides of the first strand may be modified and the even numbered nucleotides may be modified with a second modification. The second strand may comprise adjacent nucleotides that are modified with a common modification, which may be the same as the modification of the odd-numbered nucleotides of the first strand. One or more nucleotides of the second strand may also be modified with the second modification. One or more nucleotides with the second modification may be adjacent to each other and to nucleotides having a modification that is the same as the modification of the odd-numbered nucleotides of the first strand. The first strand may also comprise phosphorothioate linkages between the two nucleotides at the 3′ end and at the 5′ end or a phosphorodithioate linkage between the two nucleotides at the 3′ end. The second strand may comprise a phosphorothioate or phosphorodithioate linkage between the two nucleotides at the 5′ end. The second strand may also be conjugated to a ligand at the 5′ end.

The nucleic acid of the invention may comprise a first strand comprising adjacent nucleotides that are modified with a common modification. One or more such nucleotides may be adjacent to one or more nucleotides which may be modified with a second modification. One or more nucleotides with the second modification may be adjacent. The second strand may comprise adjacent nucleotides that are modified with a common modification, which may be the same as one of the modifications of one or more nucleotides of the first strand. One or more nucleotides of the second strand may also be modified with the second modification. One or more nucleotides with the second modification may be adjacent. The first strand may also comprise phosphorothioate linkages between the two nucleotides at the 3′ end and at the 5′ end or a phosphorodithioate linkage between the two nucleotides at the 3′ end. The second strand may comprise a phosphorothioate or phosphorodithioate linkage between the two nucleotides at the 3′ end. The second strand may also be conjugated to a ligand at the 5′ end.

The nucleotides numbered from 5′ to 3′ on the first strand and 3′ to 5′ on the second strand, 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 and 25 may be modified by a modification on the first strand. The nucleotides numbered 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24 may be modified by a second modification on the first strand. The nucleotides numbered 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 may be modified by a modification on the second strand. The nucleotides numbered 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24 may be modified by a second modification on the second strand. Nucleotides are numbered for the sake of the nucleic acid of the present invention from 5′ to 3′ on the first strand and 3′ to 5′ on the second strand.

The nucleotides numbered 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24 may be modified by a modification on the first strand. The nucleotides numbered 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 may be modified by a second modification on the first strand. The nucleotides numbered 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23 may be modified by a modification on the second strand. The nucleotides numbered 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 and 24 may be modified by a second modification on the second strand.

Clearly, if the first and/or the second strand are shorter than 25 nucleotides in length, such as 19 nucleotides in length, there are no nucleotides numbered 20, 21, 22, 23, 24 and 25 to be modified. The skilled person understands the description above to apply to shorter strands, accordingly.

One or more modified nucleotides on the first strand may be paired with modified nucleotides on the second strand having a common modification. One or more modified nucleotides on the first strand may be paired with modified nucleotides on the second strand having a different modification. One or more modified nucleotides on the first strand may be paired with unmodified nucleotides on the second strand. One or more modified nucleotides on the second strand may be paired with unmodified nucleotides on the first strand. In other words, the alternating nucleotides can be aligned on the two strands such as, for example, all the modifications in the alternating regions of the second strand are paired with identical modifications in the first strand or alternatively the modifications can be offset by one nucleotide with the common modifications in the alternating regions of one strand pairing with dissimilar modifications (i.e. a second or further modification) in the other strand. Another option is to have dissimilar modifications in each of the strands.

The modifications on the first strand may be shifted by one nucleotide relative to the modified nucleotides on the second strand, such that common modified nucleotides are not paired with each other.

The modification and/or modifications may each and individually be selected from the group consisting of 3′ terminal deoxy thymine, 2′-OMe, a 2′ deoxy modification, a 2′ amino modification, a 2′ alkyl modification, a morpholino modification, a phosphoramidate modification, 5′-phosphorothioate group modification, a 5′ phosphate or 5′ phosphate mimic modification and a cholesteryl derivative or a dodecanoic acid bisdecylamide group modification and/or the modified nucleotide may be any one of a locked nucleotide, an abasic nucleotide or a non-natural base comprising nucleotide.

At least one modification may be 2′-OMe and/or at least one modification may be 2′-F. Further modifications as described herein may be present on the first and/or second strand.

The nucleic acid of the invention may comprise an inverted RNA nucleotide at one or several of the strand ends. Such inverted nucleotides provide stability to the nucleic acid. Preferably, the nucleic acid comprises at least an inverted nucleotide at the 3′ end of the first and/or the second strand and/or at the 5′ end of the second strand. More preferably, the nucleic acid comprises an inverted nucleotide at the 3′ end of the second strand. Most preferably, the nucleic acid comprises an inverted RNA nucleotide at the 3′ end of the second strand and this nucleotide is preferably an inverted A. An inverted nucleotide is a nucleotide that is linked to the 3′ end of a nucleic acid through its 3′ carbon, rather than its 5′ carbon as would normally be the case or is linked to the 5′ end of a nucleic acid through its 5′ carbon, rather than its 3′ carbon as would normally be the case. The inverted nucleotide is preferably present at an end of a strand not as an overhang but opposite a corresponding nucleotide in the other strand. Accordingly, the nucleic acid is preferably blunt-ended at the end that comprises the inverted RNA nucleotide. An inverted RNA nucleotide being present at the end of a strand preferably means that the last nucleotide at this end of the strand is the inverted RNA nucleotide. A nucleic acid with such a nucleotide is stable and easy to synthesise. The inverted RNA nucleotide is preferably an unmodified nucleotide in the sense that it does not comprise any modifications compared to the natural nucleotide counterpart. Specifically, the inverted RNA nucleotide is preferably a 2′-OH nucleotide.

Nucleic acids of the invention may comprise one or more nucleotides modified at the 2′ position with a 2′-H, and therefore having a DNA nucleotide within the nucleic acid. Nucleic acids of the invention may comprise DNA nucleotides at positions 2 and/or 14 of the first strand counting from the 5′ end of the first strand. Nucleic acids may comprise DNA nucleotides on the second strand which correspond to position 11, or 13, or 11 and 13, or 11-13 of the first strand.

In one aspect there is no more than one DNA nucleotide per nucleic acid of the invention.

Nucleic acids of the invention may comprise one or more LNA nucleotides. Nucleic acids of the invention may comprise LNA nucleotides at positions 2 and/or 14 of the first strand counting from the 5′ end of the first strand. Nucleic acids may comprise LNA on the second strand which correspond to position 11, or 13, or 11 and 13, or 11-13 of the first strand.

Some representative modified nucleic acid sequences of the present invention are shown in the examples. These examples are meant to be representative and not limiting.

Preferably, the nucleic acid may comprise a first modification and a second or further modification which are each and individually selected from the group comprising 2′-OMe modification and 2′-F modification. The nucleic acid may comprise a modification that is 2′-OMe that may be a first modification, and a second modification that is 2′-F. The nucleic acid of the invention may also include a phosphorothioate or phosphorodithioate modification and/or a deoxy modification which may be present in or between the terminal 2 or 3 nucleotides of each or any end of each or both strands.

In one aspect of the nucleic acid, at least one nucleotide of the first and/or second strand is a modified nucleotide, wherein if the first strand comprises at least one modified nucleotide:

-   (i) at least one or both of the nucleotides 2 and 14 of the first     strand is/are modified by a first modification; and/or -   (ii) at least one, several, or all the even-numbered nucleotides of     the first strand is/are modified by a first modification; and/or -   (iii) at least one, several, or all the odd-numbered nucleotides of     the first strand is/are modified by a second modification; and/or     -   wherein if the second strand comprises at least one modified         nucleotide: -   (iv) at least one, several, or all the nucleotides of the second     strand in a position corresponding to an even-numbered nucleotide of     the first strand is/are modified by a third modification; and/or -   (v) at least one, several, or all the nucleotides of the second     strand in a position corresponding to an odd-numbered nucleotide of     the first strand is/are modified by a fourth modification; and/or -   (vi) at least one, several, or all the nucleotides of the second     strand in a position corresponding to nucleotide 11 or nucleotide 13     or nucleotides 11 and 13 or nucleotides 11-13 of the first strand     is/are modified by a fourth modification; and/or -   (vii) at least one, several, or all the nucleotides of the second     strand in a position other than the position corresponding to     nucleotide 11 or nucleotide 13 or nucleotides 11 and 13 or     nucleotides 11-13 of the first strand is/are modified by a third     modification;     -   wherein the nucleotides on the first strand are numbered         consecutively starting with nucleotide number 1 at the 5′ end of         the first strand;     -   wherein the modifications are preferably at least one of the         following:         -   (a) the first modification is preferably different from the             second and from the third modification;         -   (b) the first modification is preferably the same as the             fourth modification;         -   (c) the second and the third modification are preferably the             same modification;         -   (d) the first modification is preferably a 2′-F             modification;         -   (e) the second modification is preferably a 2′-OMe             modification;         -   (f) the third modification is preferably a 2′-OMe             modification; and/or         -   (g) the fourth modification is preferably a 2′-F             modification; and     -   wherein optionally the nucleic acid is conjugated to a ligand.

One aspect is a double-stranded nucleic acid for inhibiting expression of CNNM4, preferably in a cell, wherein the nucleic acid comprises a first strand and a second strand, wherein the first strand sequence comprises a sequence of at least 15 nucleotides differing by no more than 3 nucleotides from any one of the sequences selected from SEQ ID NO: 243, 267, 277, 279, 287, 317, 319, 325, 333, 345, 347, 349, 361, 367, 369, 371, 377, 401, 411, 413, 415, 420, 421, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 549, 550, 551 and 552, wherein all the even-numbered nucleotides of the first strand are modified by a first modification, all the odd-numbered nucleotides of the first strand are modified by a second modification, all the nucleotides of the second strand in a position corresponding to an even-numbered nucleotide of the first strand are modified by a third modification, all the nucleotides of the second strand in a position corresponding to an odd-numbered nucleotide of the first strand are modified by a fourth modification, wherein the first and fourth modification are 2′-F and the second and third modification are 2′-OMe.

One aspect is a double-stranded nucleic acid for inhibiting expression of CNNM4, preferably in a cell, wherein the nucleic acid comprises a first strand and a second strand, wherein the first strand sequence comprises a sequence of at least 15 nucleotides differing by no more than 3 nucleotides from any one of the sequences selected from SEQ ID NO: 243, 267, 277, 279, 287, 317, 319, 325, 333, 345, 347, 349, 361, 367, 369, 371, 377, 401, 411, 413, 415, 420, 421, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 549, 550, 551 and 552, wherein all the even-numbered nucleotides of the first strand are modified by a first modification, all the odd-numbered nucleotides of the first strand are modified by a second modification, all the nucleotides of the second strand in positions corresponding to nucleotides 11-13 of the first strand are modified by a fourth modification, all the nucleotides of the second strand other than the nucleotides corresponding to nucleotides 11-13 of the first strand are modified by a third modification, wherein the first and fourth modification are 2′-F and the second and third modification are 2′-OMe.

The 3′ and 5′ ends of an oligonucleotide can be modified. Such modifications can be at the 3′ end or the 5′ end or both ends of the molecule. They can include modification or replacement of an entire terminal phosphate or of one or more of the atoms of the phosphate group. For example, the 3′ and 5′ ends of an oligonucleotide can be conjugated to other functional molecular entities such as labelling moieties, e.g., fluorophores (e.g., pyrene, TAMRA, fluorescein, Cy3 or Cy5 dyes) or protecting groups (based e.g., on sulfur, silicon, boron or ester). The functional molecular entities can be attached to the sugar through a phosphate group and/or a linker. The terminal atom of the linker can connect to or replace the linking atom of the phosphate group or the C-3′ or C-5′ O, N, S or C group of the sugar. Alternatively, the linker can connect to or replace the terminal atom of a nucleotide surrogate (e.g., PNAs). These spacers or linkers can include e.g., —(CH₂)_(n)—, —(CH₂)_(n)N—, — (CH₂)_(n)O—, —(CH₂)_(n)S—, —(CH₂CH₂O)_(n)CH₂CH₂O— (e.g., n=3 or 6), abasic sugars, amide, carboxy, amine, oxyamine, oxyimine, thioether, disulfide, thiourea, sulfonamide, or morpholino, or biotin and fluorescein reagents. The 3′ end can be an —OH group.

Other examples of terminal modifications include dyes, intercalating agents (e.g., acridines), cross-linkers (e.g., psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases, EDTA, lipophilic carriers (e.g., cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g., biotin), transport/absorption facilitators (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles).

Terminal modifications can also be useful for monitoring distribution, and in such cases the groups to be added may include fluorophores, e.g., fluorescein or an Alexa dye. Terminal modifications can also be useful for enhancing uptake, useful modifications for this include cholesterol. Terminal modifications can also be useful for cross-linking an RNA agent to another moiety.

Terminal modifications can be added for a number of reasons, including to modulate activity or to modulate resistance to degradation. Terminal modifications useful for modulating activity include modification of the 5′ end with phosphate or phosphate analogues. Nucleic acids of the invention, on the first or second strand, may be 5′ phosphorylated or include a phosphoryl analogue at the 5′ prime terminus. 5′-phosphate modifications include those which are compatible with RISC mediated gene silencing. Suitable modifications include: 5′-monophosphate ((HO)₂(O)P—O-5′); 5′-diphosphate ((HO)₂(O)P—O—P(HO)(O)—O-5′); 5′-triphosphate ((HO)₂(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-guanosine cap (7-methylated or non-methylated) (7m-G-O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-adenosine cap (Appp), and any modified or unmodified nucleotide cap structure (N-O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-monothiophosphate (phosphorothioate; (HO)₂(S)P—O-5′); 5′-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P—O-5′), 5′-phosphorothiolate ((HO)₂(O)P—S-5′); any additional combination of oxygen/sulfur replaced monophosphate, diphosphate and triphosphates (e.g., 5′-alpha-thiotriphosphate, 5′-gamma-thiotriphosphate, etc.), 5′-phosphoramidates ((HO)₂(O)P—NH-5′, (HO)(NH₂)(O)P—O-5′), 5′-alkylphosphonates (alkyl=methyl, ethyl, isopropyl, propyl, etc., e.g. RP(OH)(O)—O-5′-(wherein R is an alkyl), (OH)₂(O)P-5′-CH₂-), 5′ vinylphosphonate, 5′-alkyletherphosphonates (alkylether=methoxymethyl (MeOCH₂-), ethoxymethyl, etc., e.g. RP(OH)(O)—O-5′- (wherein R is an alkylether)).

Certain moieties may be linked to the 5′ terminus of the first strand or the second strand. These include abasic ribose moiety, abasic deoxyribose moiety, modifications abasic ribose and abasic deoxyribose moieties including 2′-O alkyl modifications; inverted abasic ribose and abasic deoxyribose moieties and modifications thereof, C6-imino-Pi; a mirror nucleotide including L-DNA and L-RNA; 5′OMe nucleotide; and nucleotide analogues including 4′,5′-methylene nucleotide; 1-(β-D-erythrofuranosyl)nucleotide; 4′-thio nucleotide, carbocyclic nucleotide; 5′-amino-alkyl phosphate; 1,3-diamino-2-propyl phosphate, 3-aminopropyl phosphate; 6-aminohexyl phosphate; 12-aminododecyl phosphate; hydroxypropyl phosphate; 1,5-anhydrohexitol nucleotide; alpha-nucleotide; threo-pentofuranosyl nucleotide; acyclic 3′,4′-seco nucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentyl nucleotide, 5′-5′-inverted abasic moiety; 1,4-butanediol phosphate; 5′-amino; and bridging or non-bridging methylphosphonate and 5′-mercapto moieties.

In each sequence described herein, a C-terminal “—OH″ moiety may be substituted for a C-terminal “—NH₂” moiety, and vice-versa.

The invention also provides a nucleic acid according to any aspect of the invention described herein, wherein the first strand has a terminal 5′ (E)-vinylphosphonate nucleotide at its 5′ end. This terminal 5′ (E)-vinylphosphonate nucleotide is preferably linked to the second nucleotide in the first strand by a phosphodiester linkage.

The first strand of the nucleic acid may comprise formula (I):

where ‘(vp)-’ is the 5′ (E)-vinylphosphonate, ‘N’ is a nucleotide, ‘po’ is a phosphodiester linkage, and n is from 1 to (the total number of nucleotides in the first strand - 2), preferably wherein n is from 1 to (the total number of nucleotides in the first strand -3), more preferably wherein n is from 1 to (the total number of nucleotides in the first strand -4).

Preferably, the terminal 5′ (E)-vinylphosphonate nucleotide is an RNA nucleotide, preferably a (vp)-U.

A terminal 5′ (E)-vinylphosphonate nucleotide is a nucleotide wherein the natural phosphate group at the 5′-end has been replaced with a E-vinylphosphonate, in which the bridging 5′-oxygen atom of the terminal nucleotide of the 5′ phosphorylated strand is replaced with a methynyl (—CH═) group:

A 5′ (E)-vinylphosphonate is a 5′ phosphate mimic. A biological mimic is a molecule that is capable of carrying out the same function as and is structurally very similar to the original molecule that is being mimicked. In the context of the present invention, 5′ (E)-vinylphosphonate mimics the function of a normal 5′ phosphate, e.g. enabling efficient RISC loading. In addition, because of its slightly altered structure, 5′ (E) vinylphosphonate is capable of stabilizing the 5′-end nucleotide by protecting it from dephosphorylation by enzymes such as phosphatases.

In one aspect, the first strand has a terminal 5′ (E)-vinylphosphonate nucleotide at its 5′ end, the terminal 5′ (E)-vinylphosphonate nucleotide is linked to the second nucleotide in the first strand by a phosphodiester linkage and the first strand comprises a) more than 1 phosphodiester linkage; b) phosphodiester linkages between at least the terminal three 5′ nucleotides and/or c) phosphodiester linkages between at least the terminal four 5′ nucleotides.

In one aspect, the first strand and/or the second strand of the nucleic acid comprises at least one phosphorothioate (ps) and/or at least one phosphorodithioate (ps2) linkage between two nucleotides.

In one aspect, the first strand and/or the second strand of the nucleic acid comprises more than one phosphorothioate and/or more than one phosphorodithioate linkage.

In one aspect, the first strand and/or the second strand of the nucleic acid comprises a phosphorothioate or phosphorodithioate linkage between the terminal two 3′ nucleotides or phosphorothioate or phosphorodithioate linkages between the terminal three 3′ nucleotides.

Preferably, the linkages between the other nucleotides in the first strand and/or the second strand are phosphodiester linkages.

In one aspect, the first strand and/or the second strand of the nucleic acid comprises a phosphorothioate linkage between the terminal two 5′ nucleotides or a phosphorothioate linkages between the terminal three 5′ nucleotides.

In one aspect, the nucleic acid of the present invention comprises one or more phosphorothioate or phosphorodithioate modifications on one or more of the terminal ends of the first and/or the second strand. Optionally, each or either end of the first strand may comprise one or two or three phosphorothioate or phosphorodithioate modified nucleotides (internucleoside linkage). Optionally, each or either end of the second strand may comprise one or two or three phosphorothioate or phosphorodithioate modified nucleotides (internucleoside linkage).

In one aspect, the nucleic acid comprises a phosphorothioate linkage between the terminal two or three 3′ nucleotides and/or 5′ nucleotides of the first and/or the second strand. Preferably, the nucleic acid comprises a phosphorothioate linkage between each of the terminal three 3′ nucleotides and the terminal three 5′ nucleotides of the first strand and of the second strand. Preferably, all remaining linkages between nucleotides of the first and/or of the second strand are phosphodiester linkages.

In one aspect, the nucleic acid comprises a phosphorodithioate linkage between each of the two, three or four terminal nucleotides at the 3′ end of the first strand and/or comprises a phosphorodithioate linkage between each of the two, three or four terminal nucleotides at the 3′ end of the second strand and/or a phosphorodithioate linkage between each of the two, three or four terminal nucleotides at the 5′ end of the second strand and comprises a linkage other than a phosphorodithioate linkage between the two, three or four terminal nucleotides at the 5′ end of the first strand.

In one aspect, the nucleic acid comprises a phosphorothioate linkage between the terminal three 3′ nucleotides and the terminal three 5′ nucleotides of the first strand and of the second strand. Preferably, all remaining linkages between nucleotides of the first and/or of the second strand are phosphodiester linkages.

In one aspect, the nucleic acid:

-   (i) has a phosphorothioate linkage between the terminal three 3′     nucleotides and the terminal three 5′ nucleotides of the first     strand; -   (ii) is conjugated to a triantennary ligand either on the 3′ end     nucleotide or on the 5′ end nucleotide of the second strand; -   (iii) has a phosphorothioate linkage between the terminal three     nucleotides of the second strand at the end opposite to the one     conjugated to the triantennary ligand; and -   (iv) optionally all remaining linkages between nucleotides of the     first and/or of the second strand are phosphodiester linkages.

In one aspect, the nucleic acid:

-   (i) has a terminal 5′ (E)-vinylphosphonate nucleotide at the 5′ end     of the first strand; -   (ii) has a phosphorothioate linkage between the terminal three 3′     nucleotides on the first and second strand and between the terminal     three 5′ nucleotides on the second strand or it has a     phosphorodithioate linkage between the terminal two 3′ nucleotides     on the first and second strand and between the terminal two 5′     nucleotides on the second strand; and -   (iii) optionally all remaining linkages between nucleotides of the     first and/or of the second strand are phosphodiester linkages.

The use of a phosphorodithioate linkage in the nucleic acid of the invention reduces the variation in the stereochemistry of a population of nucleic acid molecules compared to molecules comprising a phosphorothioate in that same position. Phosphorothioate linkages introduce chiral centres and it is difficult to control which non-linking oxygen is substituted for sulphur. The use of a phosphorodithioate ensures that no chiral centre exists in that linkage and thus reduces or eliminates any variation in the population of nucleic acid molecules, depending on the number of phosphorodithioate and phosphorothioate linkages used in the nucleic acid molecule.

In one aspect, the nucleic acid comprises a phosphorodithioate linkage between the two terminal nucleotides at the 3′ end of the first strand and a phosphorodithioate linkage between the two terminal nucleotides at the 3′ end of the second strand and a phosphorodithioate linkage between the two terminal nucleotides at the 5′ end of the second strand and comprises a linkage other than a phosphorodithioate linkage between the two, three or four terminal nucleotides at the 5′ end of the first strand. Preferably, the first strand has a terminal 5′ (E)-vinylphosphonate nucleotide at its 5′ end. This terminal 5′ (E)-vinylphosphonate nucleotide is preferably linked to the second nucleotide in the first strand by a phosphodiester linkage. Preferably, all the linkages between the nucleotides of both strands other than the linkage between the two terminal nucleotides at the 3′ end of the first strand and the linkages between the two terminal nucleotides at the 3′ end and at the 5′ end of the second strand are phosphodiester linkages.

In one aspect, the nucleic acid comprises a phosphorothioate linkage between each of the three terminal 3′ nucleotides and/or between each of the three terminal 5′ nucleotides on the first strand, and/or between each of the three terminal 3′ nucleotides and/or between each of the three terminal 5′ nucleotides of the second strand when there is no phosphorodithioate linkage present at that end. No phosphorodithioate linkage being present at an end means that the linkage between the two terminal nucleotides, or preferably between the three terminal nucleotides of the nucleic acid end in question are linkages other than phosphorodithioate linkages.

In one aspect, all the linkages of the nucleic acid between the nucleotides of both strands other than the linkage between the two terminal nucleotides at the 3′ end of the first strand and the linkages between the two terminal nucleotides at the 3′ end and at the 5′ end of the second strand are phosphodiester linkages.

Other phosphate linkage modifications are possible.

The phosphate linker can also be modified by replacement of a linking oxygen with nitrogen (bridged phosphoroamidates), sulfur (bridged phosphorothioates) and carbon (bridged methylenephosphonates). The replacement can occur at a terminal oxygen. Replacement of the non-linking oxygens with nitrogen is possible.

The phosphate groups can also individually be replaced by non-phosphorus containing connectors.

Examples of moieties which can replace the phosphate group include siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo and methyleneoxymethylimino. In certain embodiments, replacements may include the methylenecarbonylamino and methylenemethylimino groups.

The phosphate linker and ribose sugar may be replaced by nuclease resistant nucleotides. Examples include the morpholino, cyclobutyl, pyrrolidine and peptide nucleic acid (PNA) nucleoside surrogates. In certain embodiments, PNA surrogates may be used.

In one aspect, the nucleic acid, which is preferably an siRNA that inhibits expression of CNNM4, preferably via RNAi, and preferably in a cell, comprises one or more or all of:

-   (i) a modified nucleotide; -   (ii) a modified nucleotide other than a 2′-OMe modified nucleotide     at positions 2 and 14 from the 5′ end of the first strand,     preferably a 2′-F modified nucleotide; -   (iii) each of the odd-numbered nucleotides of the first strand as     numbered starting from one at the 5′ end of the first strand are     2′-OMe modified nucleotides; -   (iv) each of the even-numbered nucleotides of the first strand as     numbered starting from one at the 5′ end of the first strand are     2′-F modified nucleotides; -   (v) the second strand nucleotide corresponding to position 11 and/or     13 or 11-13 of the first strand is modified by a modification other     than a 2′-OMe modification, preferably wherein one or both or all of     these positions comprise a 2′-F modification; -   (vi) an inverted nucleotide, preferably a 3′-3′ linkage at the 3′     end of the second strand; -   (vii) one or more phosphorothioate linkages; -   (viii) one or more phosphorodithioate linkages; and/or -   (ix) the first strand has a terminal 5′ (E)-vinylphosphonate     nucleotide at its 5′ end, in which case the terminal 5′     (E)-vinylphosphonate nucleotide is preferably a uridine and is     preferably linked to the second nucleotide in the first strand by a     phosphodiester linkage.

All the features of the nucleic acids can be combined with all other aspects of the invention disclosed herein.

Ligands

The nucleic acids of the invention may be conjugated to a ligand. Efficient delivery of oligonucleotides, in particular double-stranded nucleic acids of the invention, to cells in vivo is important and requires specific targeting and substantial protection from the extracellular environment, particularly serum proteins. One method of achieving specific targeting is to conjugate a ligand to the nucleic acid. In some embodiments, the ligand helps in targeting the nucleic acid to a target cell which has a cell surface receptor that binds to and internalizes the conjugated ligand. In such embodiments, there is a need to conjugate appropriate ligands for the desired receptor molecules in order for the conjugated molecules to be taken up by the target cells by mechanisms such as different receptor-mediated endocytosis pathways or functionally analogous processes. In other embodiments, a ligand which can mediate internalization of the nucleic acid into a target cell by mechanisms other than receptor mediated endocytosis may alternatively be conjugated to a nucleic acid of the invention for cell or tissue specific targeting.

One example of a conjugate that mediates receptor mediated endocytosis is the asialoglycoprotein receptor complex (ASGP-R) which has high affinity to the GalNAc moiety described herein. The ASGP-R complex is composed of varying ratios of multimers of membrane ASGR1 and ASGR2 receptors, which are highly abundant on hepatocytes. One of the first disclosures of the use of triantennary cluster glycosides as conjugated ligands was in US patent number US 5,885,968. Conjugates having three GalNAc ligands and comprising phosphate groups are known and are described in Dubber et al. (Bioconjug. Chem. 2003 Jan-Feb;14(1):239-46.). The ASGP-R complex shows a 50-fold higher affinity for N-Acetyl-D-Galactosamine (GalNAc) than D-Gal.

The ASGP-R complex recognizes specifically terminal β-galactosyl subunits of glycosylated proteins or other oligosaccharides (Weigel, P.H. et. al., Biochim. Biophys. Acta. 2002 Sep 19;1572(2-3):341-63) and can be used for delivering a drug to the liver’s hepatocytes expressing the receptor complex by covalent coupling of galactose or galactosamine to the drug substance (Ishibashi,S.; et. al., J Biol. Chem. 1994 Nov 11;269(45):27803-6). Furthermore, the binding affinity can be significantly increased by the multi-valency effect, which is achieved by the repetition of the targeting moiety (Biessen EA, et al., J Med Chem. 1995 Apr 28;38(9):1538-46).

The ASGP-R complex is a mediator for an active uptake of terminal β-galactosyl containing glycoproteins to the cell’s endosomes. Thus, the ASGPR is highly suitable for targeted delivery of drug candidates conjugated to such ligands like, e.g., nucleic acids into receptor-expressing cells (Akinc et al., Mol Ther. 2010 Jul;18(7):1357-64).

More generally the ligand can comprise a saccharide that is selected to have an affinity for at least one type of receptor on a target cell. In particular, the receptor is on the surface of a mammalian liver cell, for example, the hepatic asialoglycoprotein receptor complex described before (ASGP-R).

The saccharide may be selected from N-acetyl galactosamine, mannose, galactose, glucose, glucosamine and fucose. The saccharide may be N-acetyl galactosamine (GalNAc).

A ligand for use in the present invention may therefore comprise (i) one or more N-acetyl galactosamine (GalNAc) moieties and derivatives thereof, and (ii) a linker, wherein the linker conjugates the GalNAc moieties to a nucleic acid as defined in any preceding aspects. The linker may be a monovalent structure or bivalent or trivalent or tetravalent branched structure. The nucleotides may be modified as defined herein.

The ligand may therefore comprise GalNAc.

In one aspect, the nucleic acid is conjugated to a ligand comprising a compound of formula (II):

wherein:

-   S represents a saccharide, preferably wherein the saccharide is     N-acetyl galactosamine; -   X¹ represents C₃-C₆ alkylene or (—CH₂—CH₂—O)_(m)(—CH₂)₂— wherein m     is 1, 2, or 3; -   P is a phosphate or modified phosphate, preferably a thiophosphate; -   X² is alkylene or an alkylene ether of the formula (—CH₂)_(n)—O—CH₂—     where n = 1-6; -   A is a branching unit; -   X³ represents a bridging unit; -   wherein a nucleic acid according to the present invention is     conjugated to X³ via a phosphate or modified phosphate, preferably a     thiophosphate.

In formula (II), the branching unit “A” preferably branches into three in order to accommodate three saccharide ligands. The branching unit is preferably covalently attached to the remaining tethered portions of the ligand and the nucleic acid. The branching unit may comprise a branched aliphatic group comprising groups selected from alkyl, amide, disulphide, polyethylene glycol, ether, thioether and hydroxyamino groups. The branching unit may comprise groups selected from alkyl and ether groups.

The branching unit A may have a structure selected from:

wherein each A₁ independently represents O, S, C═O or NH; and each n independently represents an integer from 1 to 20.

The branching unit may have a structure selected from:

wherein each A₁ independently represents O, S, C═O or NH; and each n independently represents an integer from 1 to 20.

The branching unit may have a structure selected from:

wherein A₁ is O, S, C═O or NH; and each n independently represents an integer from 1 to 20.

The branching unit may have the structure:

The branching unit may have the structure:

indicates text missing or illegible when filed

The branching unit may have the structure:

Alternatively, the branching unit A may have a structure selected from:

wherein:

-   R1 is hydrogen or C1-C10 alkylene; -   and R2 is C1-C10 alkylene.

Optionally, the branching unit consists of only a carbon atom.

The “X³” portion is a bridging unit. The bridging unit is linear and is covalently bound to the branching unit and the nucleic acid.

X³ may be selected from -C₁-C₂₀ alkylene-, -C₂-C₂₀ alkenylene-, an alkylene ether of formula -(C₁-C₂₀ alkylene)―O―(C₁-C₂₀ alkylene)-, -C(O)-C₁-C₂₀ alkylene-, -C₀-C₄ alkylene(Cy)C₀-C₄ alkylene- wherein Cy represents a substituted or unsubstituted 5 or 6 membered cycloalkylene, arylene, heterocyclylene or heteroarylene ring, -C₁-C₄ alkylene-NHC(O)-C₁-C₄ alkylene-, -C₁-C₄ alkylene-C(O)NH-C₁-C₄ alkylene-, -C₁-C₄ alkylene-SC(O)-C₁-C₄ alkylene-, -C₁-C₄ alkylene-C(O)S-C₁-C₄ alkylene-, -C₁-C₄ alkylene-OC(O)-C₁-C₄ alkylene-, -C₁-C₄ alkylene-C(O)O-C₁-C₄ alkylene-, and -C₁-C₆ alkylene-S-S-C₁-C₆ alkylene-.

X³ may be an alkylene ether of formula -(C₁-C₂₀ alkylene)―O―(C₁-C₂₀ alkylene)-. X³ may be an alkylene ether of formula -(C₁-C₂₀ alkylene)―O―(C₄-C₂₀ alkylene)-, wherein said (C₄-C₂₀ alkylene) is linked to Z. X³ may be selected from the group consisting of —CH₂—O—C₃H₆—, —CH₂—O—C₄H₈—, —CH₂—O—C₆H₁₂— and —CH₂—O—C₈H₁₆—, especially —CH₂—O—C₄H₈—, —CH₂—O—C₆H₁₂— and —CH₂—O—C₈H₁₆—, wherein in each case the —CH₂— group is linked to A.

In one aspect, the nucleic acid is conjugated to a ligand comprising a compound of formula (III):

wherein:

-   S represents a saccharide, preferably GalNAc;

-   X¹ represents C₃-C₆ alkylene or (—CH₂—CH₂—O)_(m)(—CH₂)₂— wherein m     is 1, 2, or 3;

-   P is a phosphate or modified phosphate, preferably a thiophosphate;

-   X² is C₁-C₈ alkylene;

-   A is a branching unit selected from:

-   

-   

-   X³ is a bridging unit;

wherein a nucleic acid according to the present invention is conjugated to X³ via a phosphate or a modified phosphate, preferably a thiophosphate.

The branching unit A may have the structure:

The branching unit A may have the structure:

wherein X³ is attached to the nitrogen atom.

X³ may be C₁-C₂₀ alkylene. Preferably, X³ is selected from the group consisting of —C₃H₆—, —C₄H₈—,—C₆H₁₂— and —C₈H₁₆—, especially —C₄H₈—,—C₆H₁₂— and —C₈H₁₆—.

In one aspect, the nucleic acid is conjugated to a ligand comprising a compound of formula (IV):

wherein:

-   S represents a saccharide, preferably GalNAc; -   X¹ represents C₃-C₆ alkylene or (—CH₂—CH₂—O)_(m)(—CH₂)₂— wherein m     is 1, 2, or 3; -   P is a phosphate or modified phosphate, preferably a thiophosphate; -   X² is an alkylene ether of formula —C₃H₆—O—CH₂—; -   A is a branching unit; -   X³ is an alkylene ether of formula selected from the group     consisting of —CH₂—O—CH₂—, —CH₂—O—C₂H₄—, —CH₂—O—C₃H₆—, —CH₂—O—C₄H₈—,     —CH₂—O—C₅H₁₀—, —CH₂—O—C₆H₁₂—, —CH₂—O—C₇H₁₄—, and —CH₂—O—C₈H₁₆—,     wherein in each case the —CH₂— group is linked to A, -   and wherein X³ is conjugated to a nucleic acid according to the     present invention by a phosphate or modified phosphate, preferably a     thiophosphate.

The branching unit may comprise carbon. Preferably, the branching unit is a carbon.

X³ may be selected from the group consisting of —CH₂—O—C₄H₈—, —CH₂—O—C₅H₁₀—, —CH₂—O—C₆H₁₂—, —CH₂—O—C₇H₁₄—, and —CH₂—O—C₈H₁₆—. Preferably, X³ is selected from the group consisting of —CH₂—O—C₄H₈—, —CH₂—O—C₆H₁₂— and —CH₂—O—C₈H₁₆.

X¹ may be (—CH₂—CH₂—O)(—CH₂)₂—. X¹ may be (—CH₂—CH₂—O)₂(—CH₂)₂—. X¹ may be (—CH₂—CH₂—O)₃(—CH₂)₂—. Preferably, X¹ is (—CH₂—CH₂—O)₂(—CH₂)₂—. Alternatively, X¹ represents C₃-C₆ alkylene. X¹ may be propylene. X¹ may be butylene. X¹ may be pentylene. X¹ may be hexylene. Preferably the alkyl is a linear alkylene. In particular, X¹ may be butylene.

X² represents an alkylene ether of formula —C₃H₆—O—CH₂— i.e. C₃ alkoxy methylene, or —CH₂CH₂CH₂OCH₂—.

For any of the above aspects, when P represents a modified phosphate group, P can be represented by:

wherein Y¹ and Y² each independently represent =O, =S, —O⁻, —OH, —SH, —BH₃, —OCH₂CO₂, -OCH₂CO₂R^(x), -OCH₂C(S)OR^(x), and -OR^(x), wherein R^(x) represents C₁-C₆ alkyl and wherein

indicates attachment to the remainder of the compound.

By modified phosphate it is meant a phosphate group wherein one or more of the non-linking oxygens is replaced. Examples of modified phosphate groups include phosphorothioate, phosphorodithioates, phosphoroselenates, borano phosphates, borano phosphate esters, hydrogen phosphonates, phosphoroamidates, alkyl or aryl phosphonates and phosphotriesters. Phosphorodithioates have both non-linking oxygens replaced by sulphur. One, each or both non-linking oxygens in the phosphate group can be independently any one of S, Se, B, C, H, N, or OR (R is alkyl or aryl).

The phosphate can also be modified by replacement of a linking oxygen with nitrogen (bridged phosphoroamidates), sulfur (bridged phosphorothioates) and carbon (bridged methylenephosphonates). The replacement can occur at a terminal oxygen. Replacement of the non-linking oxygens with nitrogen is possible.

For example, Y¹ may represent —OH and Y² may represent ═O or ═S; or

-   Y¹ may represent —O⁻ and Y² may represent ═O or ═S; -   Y¹ may represent ═O and Y² may represent —CH₃, —SH, —OR^(x), or —BH₃ -   Y¹ may represent =S and Y² may represent —CH₃, OR^(x) or —SH.

It will be understood by the skilled person that in certain instances there will be delocalisation between Y¹ and Y².

Preferably, the modified phosphate group is a thiophosphate group. Thiophosphate groups include bithiophosphate (i.e. where Y¹ represents ═S and Y² represents —S⁻) and monothiophosphate (i.e. where Y¹ represents —O⁻ and Y² represents ═S, or where Y¹ represents ═O and Y² represents —S⁻). Preferably, P is a monothiophosphate. The inventors have found that conjugates having thiophosphate groups in replacement of phosphate groups have improved potency and duration of action in vivo.

P may also be an ethylphosphate (i.e. where Y¹ represents ═O and Y² represents OCH₂CH₃).

The saccharide may be selected to have an affinity for at least one type of receptor on a target cell. In particular, the receptor is on the surface of a mammalian liver cell, for example, the hepatic asialoglycoprotein receptor complex (ASGP-R).

For any of the above or below aspects, the saccharide may be selected from N-acetyl with one or more of galactosamine, mannose, galactose, glucose, glucosamine and fructose. Typically a ligand to be used in the present invention may include N-acetyl galactosamine (GaINAc). Preferably the compounds of the invention may have 3 ligands, which will each preferably include N-acetyl galactosamine.

“GalNAc” refers to 2-(Acetylamino)-2-deoxy-D-galactopyranose, commonly referred to in the literature as N-acetyl galactosamine. Reference to “GalNAc” or “N-acetyl galactosamine” includes both the β- form: 2-(Acetylamino)-2-deoxy-β -D-galactopyranose and the α-form: 2-(Acetylamino)-2-deoxy-α-D- galactopyranose. In certain embodiments, both the β-form: 2-(Acetylamino)-2-deoxy-β-D-galactopyranose and α-form: 2-(Acetylamino)-2-deoxy-α-D-galactopyranose may be used interchangeably. Preferably, the compounds of the invention comprise the β-form, 2-(Acetylamino)-2-deoxy-β-D-galactopyranose.

2-(Acetylamino)-2-deoxy-D-galactopyranose

2-(Acetylamino)-2-deoxy-β-D-galactopyranose

2-(Acetylamino)-2-deoxy-α-D-galactopyranose

In one aspect, the nucleic acid is a conjugated nucleic acid, wherein the nucleic acid is conjugated to a triantennary ligand with one of the following structures:

wherein Z is any nucleic acid as defined herein.

Preferably, the nucleic acid is a conjugated nucleic acid, wherein the nucleic acid is conjugated to a triantennary ligand with the following structures:

wherein Z is any nucleic acid as defined herein.

A ligand of formula (II), (III) or (IV) or any one of the triantennary ligands disclosed herein can be attached at the 3′-end of the first (antisense) strand and/or at any of the 3′ and/or 5′ end of the second (sense) strand. The nucleic acid can comprise more than one ligand of formula (II), (III) or (IV) or any one of the triantennary ligands disclosed herein. However, a single ligand of formula (II), (III) or (IV) or any one of the triantennary ligands disclosed herein is preferred because a single such ligand is sufficient for efficient targeting of the nucleic acid to the target cells. Preferably in that case, at least the last two, preferably at least the last three and more preferably at least the last four nucleotides at the end of the nucleic acid to which the ligand is attached are linked by a phosphodiester linkage.

Preferably, the 5′-end of the first (antisense) strand is not attached to a ligand of formula (II), (III) or (IV) or any one of the triantennary ligands disclosed herein, since a ligand in this position can potentially interfere with the biological activity of the nucleic acid.

A nucleic acid with a single ligand of formula (II), (III) or (IV) or any one of the triantennary ligands disclosed herein at the 5′ end of a strand is easier and therefore cheaper to synthesise than the same nucleic acid with the same ligand at the 3′ end. Preferably therefore, a single ligand of any of formulae (II), (III) or (IV) or any one of the triantennary ligands disclosed herein is covalently attached to (conjugated with) the 5′ end of the second strand of the nucleic acid.

In one aspect, the first strand of the nucleic acid is a compound of formula (V):

wherein b is preferably 0 or 1; and

-   the second strand is a compound of formula (VI):

-   

-   wherein:     -   c and d are independently preferably 0 or 1;     -   Z₁ and Z₂ are respectively the first and second strand of the         nucleic acid;     -   Y is independently O or S;     -   n is independently 0, 1, 2 or 3; and     -   L₁ is a linker to which a ligand is attached, wherein L₁ is the         same or different in formulae (V) and (VI), and is the same or         different within formulae (V) and (VI) when L₁ is present more         than once within the same formula, wherein L₁ is preferably of         formula (VII);

    and wherein b + c + d is preferably 2 or 3.

Preferably, L₁ in formulae (V) and (VI) is of formula (VII):

wherein:

-   L is selected from the group comprising, or preferably consisting     of:     -   -(CH₂)_(r)-C(O)-, wherein r = 2-12;     -   -(CH₂-CH₂-O)_(s)-CH₂-C(O)-, wherein s = 1-5;     -   -(CH₂)_(t)-CO-NH-(CH₂)_(t)-NH-C(O)-, wherein t is independently         1-5;     -   -(CH₂)_(u)-CO-NH-(CH₂)_(u)-C(O)-, wherein u is independently         1-5; and     -   -(CH₂)_(v)-NH-C(O)-, wherein v is 2-12; and

-   wherein the terminal C(O), if present, is attached to X of formula     (VII), or if X is absent, to W₁ of formula (VII), or if W₁ is     absent, to V of formula (VII);

-   W₁, W₃ and W₅ are individually absent or selected from the group     comprising, or preferably consisting of:     -   -(CH₂)_(r)-, wherein r = 1-7;     -   -(CH₂)_(s)-O-(CH₂)_(s)-, wherein s is independently 0-5;     -   -(CH₂)_(t)-S-(CH₂)_(t)-, wherein t is independently 0-5;

-   X is absent or is selected from the group comprising, or preferably     consisting of: NH, NCH₃ or NC₂H₅;

-   V is selected from the group comprising, or preferably consisting     of:

-   

-   

-   

-   

-   

-   wherein B, if present, is a modified or natural nucleobase.

In one aspect, the first strand is a compound of formula (VIII)

-   wherein b is preferably 0 or 1; and

-   the second strand is a compound of formula (IX):

-   

-   wherein c and d are independently preferably 0 or 1; wherein:     -   Z₁ and Z₂ are respectively the first and second strand of the         nucleic acid;     -   Y is independently O or S;     -   R₁ is H or methyl;     -   n is independently preferably 0, 1, 2 or 3; and     -   L is the same or different in formulae (VIII) and (IX), and is         the same or different within formulae (VIII) and (IX) when L is         present more than once within the same formula, and is selected         from the group comprising, or preferably consisting of:         -   -(CH₂)_(r)-C(O)-, wherein r = 2-12;         -   -(CH₂-CH₂-O)_(s)-CH₂-C(O)-, wherein s = 1-5;         -   -(CH₂)_(t)-CO-NH-(CH₂)_(t)-NH-C(O)-, wherein t is             independently 1-5;         -   -(CH₂)_(u)-CO-NH-(CH₂)_(u)-C(O)-, wherein u is independently             1-5; and         -   -(CH₂)_(v)-NH-C(O)-, wherein v is 2-12; and     -   wherein the terminal C(O), if present, is attached to the NH         group (of the linker, not of the targeting ligand);

-   and wherein b + c + d is preferably 2 or 3.

In one aspect, the first strand of the nucleic acid is a compound of formula (X):

-   wherein b is preferably 0 or 1; and

-   the second strand is a compound of formula (XI):

-   

-   wherein:     -   c and d are independently preferably 0 or 1;

    -   Z₁ and Z₂ are respectively the first and second RNA strand of         the nucleic;

    -   Y is independently O or S;

    -   n is independently preferably 0, 1, 2 or 3; and

    -   L₂ is the same or different in formulae (X) and (XI) and is the         same or different in moieties bracketed by b, c and d, and is         selected from the group comprising, or preferably consisting of:

    -   

    -   

    -   

    -   n is 0 and L₂ is:

    -   

    -   and the terminal OH group is absent such that the following         moiety is formed:

    -   

    -   wherein:         -   F is a saturated branched or unbranched (such as unbranched)             C₁₋₈alkyl (e.g. C₁₋₆alkyl) chain wherein one of the carbon             atoms is optionally replaced with an oxygen atom provided             that said oxygen atom is separated from another heteroatom             (e.g. an O or N atom) by at least 2 carbon atoms;         -   L is the same or different in formulae (X) and (XI) and is             selected from the group comprising, or preferably consisting             of:             -   -(CH₂)_(r)-C(O)-, wherein r = 2-12;             -   -(CH₂-CH₂-O)_(s)-CH₂-C(O)-, wherein s = 1-5;             -   -(CH₂)_(t)-CO-NH-(CH₂)_(t)-NH-C(O)-, wherein t is                 independently 1-5;             -   -(CH₂)_(u)-CO-NH-(CH₂)_(u)-C(O)-, wherein u is                 independently 1-5; and             -   -(CH₂)_(v)-NH-C(O)-, wherein v is 2-12; and         -   wherein the terminal C(O), if present, is attached to the NH             group (of the linker, not of the targeting ligand);

-   and wherein b + c + d is preferably 2 or 3.

In one aspect, b is 0, c is 1 and d is 1; b is 1, c is 0 and d is 1; b is 1, c is 1 and d is 0; or b is 1, c is 1 and d is 1 in any of the nucleic acids of formulae (V) and (VI) or (VIII) and (IX) or (X) and (XI). Preferably, b is 0, c is 1 and d is 1; b is 1, c is 0 and d is 1; or b is 1, c is 1 and d is 1. Most preferably, b is 0, c is 1 and d is 1.

In one aspect, Y is O in any of the nucleic acids of formulae (V) and (VI) or (VIII) and (IX) or (X) and (XI). In another aspect, Y is S. In a preferred aspect, Y is independently selected from O or S in the different positions in the formulae.

In one aspect, R₁ is H or methyl in any of the nucleic acids of formulae (VIII) and (IX). In one aspect, R₁ is H. In another aspect, R₁ is methyl.

In one aspect, n is 0, 1, 2 or 3 in any of the nucleic acids of formulae (V) and (VI) or (VIII) and (IX) or (X) and (XI). Preferably, n is 0.

Examples of F moieties in any of the nucleic acids of formulae (X) and (XI) include (CH₂)₁₋₆ e.g. (CH₂)₁₋₄ e.g. CH₂, (CH₂)₄, (CH₂)₅ or (CH₂)₆, or CH₂O(CH₂)₂₋₃, e.g. CH₂O(CH₂)CH₃.

In one aspect, L₂ in formulae (X) and (XI) is:

In one aspect, L₂ is:

In one aspect, L₂ is:

In one aspect, L₂ is:

In one aspect, n is 0 and L₂ is:

and the terminal OH group is absent such that the following moiety is formed:

wherein Y is O or S.

In one aspect, L in the nucleic acids of formulae (V) and (VI) or (VIII) and (IX) or (X) and (XI), is selected from the group comprising, or preferably consisting of:

-   -(CH₂)_(r)-C(O)-, wherein r = 2-12; -   -(CH₂-CH₂-O)_(s)-CH₂-C(O)-, wherein s = 1-5; -   -(CH₂)_(t)-CO-NH-(CH₂)_(t)-NH-C(O)-, wherein t is independently 1-5; -   -(CH₂)_(u)-CO-NH-(CH₂)_(u)-C(O)-, wherein u is independently 1-5;     and -   -(CH₂)_(v)-NH-C(O)-, wherein v is 2-12;

wherein the terminal C(O) is attached to the NH group.

Preferably, L is -(CH₂)_(r)-C(O)-, wherein r = 2-12, more preferably r = 2-6 even more preferably, r = 4 or 6 e.g. 4.

Preferably, L is:

Within the moiety bracketed by b, c and d, L₂ in the nucleic acids of formulae (X) and (XI) is typically the same. Between moieties bracketed by b, c and d, L₂ may be the same or different. In an embodiment, L₂ in the moiety bracketed by c is the same as the L₂ in the moiety bracketed by d. In an embodiment, L₂ in the moiety bracketed by c is not the same as L₂ in the moiety bracketed by d. In an embodiment, the L₂ in the moieties bracketed by b, c and d is the same, for example when the linker moiety is a serinol-derived linker moiety.

Serinol derived linker moieties may be based on serinol in any stereochemistry i.e. derived from L-serine isomer, D-serine isomer, a racemic serine or other combination of isomers. In a preferred aspect of the invention, the serinol-GalNAc moiety (SerGN) has the following stereochemistry:

i.e. is based on an (S)-serinol-amidite or (S)-serinol succinate solid supported building block derived from L-serine isomer.

In a preferred aspect, the first strand of the nucleic acid is a compound of formula (VIII) and the second strand of the nucleic acid is a compound of formula (IX), wherein:

-   b is 0; -   c and d are 1, -   n is 0, -   Z₁ and Z₂ are respectively the first and second strand of the     nucleic acid, -   Y is S, -   R₁ is H, and -   L is —(CH₂)₄—C(O)—, wherein the terminal C(O) of L is attached to     the N atom of the linker (ie not a possible N atom of a targeting     ligand).

In another preferred aspect, the first strand of the nucleic acid is a compound of formula (V) and the second strand of the nucleic acid is a compound of formula (VI), wherein:

-   b is 0, -   c and d are 1, -   n is 0, -   Z₁ and Z₂ are respectively the first and second strand of the     nucleic acid, -   Y is S, -   L₁ is of formula (VII), wherein:     -   W₁ is —CH₂—O—(CH₂)₃—,     -   W₃ is —CH₂—,     -   W₅ is absent,     -   V is CH,     -   X is NH, and     -   L is —(CH₂)₄—C(O)— wherein the terminal C(O) of L is attached to         the N atom of X in formula (VII).

In another preferred aspect, the first strand of the nucleic acid is a compound of formula (V) and the second strand of the nucleic acid is a compound of formula (VI), wherein:

-   b is 0, -   c and d are 1, -   n is 0, -   Z₁ and Z₂ are respectively the first and second strand of the     nucleic acid, -   Y is S, -   L₁ is of formula (VII), wherein:     -   W₁, W₃ and W₅ are absent,

    -   

    -   X is absent, and

    -   L is —(CH₂)₄—C(O)—NH—(CH₂)₅—C(O)—, wherein the terminal C(O) of         L is attached to the N atom of V in formula (VII).

In one aspect, the nucleic acid is conjugated to a triantennary ligand with the following structure:

wherein the nucleic acid is conjugated to the ligand via the phosphate group of the ligand a) to the last nucleotide at the 5′ end of the second strand; b) to the last nucleotide at the 3′ end of the second strand; or c) to the last nucleotide at the 3′ end of the first strand.

In one aspect of the nucleic acid, the cells that are targeted by the nucleic acid with a ligand are hepatocytes.

In any one of the above ligands where GalNAc is present, the GalNAc may be substituted for any other targeting ligand, such as those mentioned herein, in particular mannose, galactose, glucose, glucosamine and fucose.

A particularly preferred embodiment is a nucleic acid wherein the first strand comprises or consists of SEQ ID NO: 237 and the second strand optionally comprises or consists of SEQ ID NO: 156. This nucleic acid can be further conjugated to a ligand, preferably at the 5′ end of the second (sense) strand. Even more preferred is a nucleic acid wherein the first strand comprises or consists of SEQ ID NO: 237 and the second strand optionally comprises or consists of SEQ ID NO: 238. Most preferred in this embodiment is a siRNA that consists of SEQ ID NO: 237 and SEQ ID NO: 238. One aspect of the invention is EU414.

An alternative particularly preferred embodiment is a nucleic acid wherein the first strand comprises or consists of SEQ ID NO: 237 and the second strand optionally comprises or consists of SEQ ID NO: 553. This nucleic acid can be further conjugated to a ligand, preferably at the 5′ end of the second (sense) strand. Even more preferred is a nucleic acid wherein the first strand comprises or consists of SEQ ID NO: 237 and the second strand optionally comprises or consists of SEQ ID NO: 460. Most preferred in this embodiment is a siRNA that consists of SEQ ID NO: 237 and SEQ ID NO: 460. One aspect of the invention is EU420.

An alternative particularly preferred embodiment is a nucleic acid wherein the first strand comprises or consists of SEQ ID NO: 461 and the second strand optionally comprises or consists of SEQ ID NO: 554. This nucleic acid can be further conjugated to a ligand, preferably at the 5′ end of the second (sense) strand. Even more preferred is a nucleic acid wherein the first strand comprises or consists of SEQ ID NO: 461 and the second strand optionally comprises or consists of SEQ ID NO: 462. Most preferred in this embodiment is a siRNA that consists of SEQ ID NO: 461 and SEQ ID NO: 462. One aspect of the invention is EU422.

In one aspect, the nucleic acid is conjugated to a ligand that comprises a lipid, and more preferably, a ligand that comprises a cholesterol.

Compositions, Uses and Methods

The present invention also provides compositions comprising a nucleic acid of the invention. The nucleic acids and compositions may be used as medicaments or as diagnostic agents, alone or in combination with other agents. For example, one or more nucleic acid(s) of the invention can be combined with a delivery vehicle (e.g., liposomes) and/or excipients, such as carriers, diluents. Other agents such as preservatives and stabilizers can also be added. Pharmaceutically acceptable salts or solvates of any of the nucleic acids of the invention are likewise within the scope of the present invention. Methods for the delivery of nucleic acids are known in the art and within the knowledge of the person skilled in the art.

Compositions disclosed herein are particularly pharmaceutical compositions. Such compositions are suitable for administration to a subject.

In one aspect, the composition comprises a nucleic acid disclosed herein, or a pharmaceutically acceptable salt or solvate thereof, and a solvent and/or a delivery vehicle and/or a physiologically acceptable excipient and/or a carrier and/or a salt and/or a diluent and/or a buffer and/or a preservative.

Pharmaceutically acceptable carriers or diluents include those used in formulations suitable for oral, rectal, nasal or parenteral (including subcutaneous, intramuscular, intravenous, intradermal, and transdermal) administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Subcutaneous or transdermal modes of administration may be particularly suitable for the compounds described herein.

The therapeutically effective amount of a nucleic acid of the present invention will depend on the route of administration, the type of mammal being treated, and the physical characteristics of the specific mammal under consideration. These factors and their relationship to determining this amount are well known to skilled practitioners in the medical arts. This amount and the method of administration can be tailored to achieve optimal efficacy, and may depend on such factors as weight, diet, concurrent medication and other factors, well known to those skilled in the medical arts. The dosage sizes and dosing regimen most appropriate for human use may be guided by the results obtained by the present invention, and may be confirmed in properly designed clinical trials.

An effective dosage and treatment protocol may be determined by conventional means, starting with a low dose in laboratory animals and then increasing the dosage while monitoring the effects, and systematically varying the dosage regimen as well. Numerous factors may be taken into consideration by a clinician when determining an optimal dosage for a given subject. Such considerations are known to the skilled person.

Nucleic acids of the present invention, or salts thereof, may be formulated as pharmaceutical compositions prepared for storage or administration, which typically comprise a therapeutically effective amount of a nucleic acid of the invention, or a salt thereof, in a pharmaceutically acceptable carrier.

The nucleic acid or conjugated nucleic acid of the present invention can also be administered in combination with other therapeutic compounds, either administrated separately or simultaneously, e.g., as a combined unit dose. The invention also includes a composition comprising one or more nucleic acids according to the present invention in a physiologically/pharmaceutically acceptable excipient, such as a stabilizer, preservative, diluent, buffer, and the like.

In one aspect, the composition comprises a nucleic acid disclosed herein and a further therapeutic agent selected from the group comprising an oligonucleotide, a small molecule, a monoclonal antibody, a polyclonal antibody and a peptide. In one embodiment, the further therapeutic agent is an inhibitor of CNNM4. In a preferred embodiment, the further therapeutic agent is the small molecule 7-amino-2-phenyl-5H-thieno[3,2-c]pyridin-4-one (PubChem CID 91383855) or the rhodanine derivative 2-[5-(4-Oxo-2-thioxo-thiazolidin-5-ylidenemethyl)-furan-2-yl]-benzoic acid as described in Park, H., et al. 2008. Bioorg Med Chem Lett. 18(7):2250-5 (Chemspider ID 1014170).

In certain embodiments, two or more nucleic acids of the invention with different sequences may be administered simultaneously or sequentially.

In another aspect, the present invention provides a composition, e.g., a pharmaceutical composition, comprising one or a combination of different nucleic acids of the invention and at least one pharmaceutically acceptable carrier.

Dosage levels for the medicament and compositions of the invention can be determined by those skilled in the art by experimentation. In one aspect, a unit dose may contain between about 0.01 mg/kg and about 100 mg/kg body weight of nucleic acid or conjugated nucleic acid. Alternatively, the dose can be from 10 mg/kg to 25 mg/kg body weight, or 1 mg/kg to 10 mg/kg body weight, or 0.05 mg/kg to 5 mg/kg body weight, or 0.1 mg/kg to 5 mg/kg body weight, or 0.1 mg/kg to1 mg/kg body weight, or 0.1 mg/kg to 0.5 mg/kg body weight, or 0.5 mg/kg to 1 mg/kg body weight. Alternatively, the dose can be from about 0.5 mg/kg to about 10 mg/kg body weight, or about 0.6 mg/kg to about 8 mg/kg body weight, or about 0.7 mg/kg to about 7 mg/kg body weight, or about 0.8 mg/kg to about 6 mg/kg body weight, or about 0.9 mg/kg to about 5.5 mg/kg body weight, or about 1 mg/kg to about 5 mg/kg body weight, or about 1 mg/kg body weight, or about 3 mg/kg body weight, or about 1 mg/kg body weight, or about 3 mg/kg body weight, or about 5 mg/kg body weight, wherein “about” is a deviation of up to 30%, preferably up to 20%, more preferably up to 10%, yet more preferably up to 5% and most preferably 0% from the indicated value. Dosage levels may also be calculated via other parameters such as, e.g., body surface area.

The dosage and frequency of administration may vary depending on whether the treatment is therapeutic or prophylactic (e.g., preventative), and may be adjusted during the course of treatment. In certain prophylactic applications, a relatively low dosage is administered at relatively infrequent intervals over a relatively long period of time. Some subjects may continue to receive treatment over their lifetime. In certain therapeutic applications, a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or until the patient shows partial or complete amelioration of symptoms of disease. Thereafter, the patient may be switched to a suitable prophylactic dosing regimen.

Actual dosage levels of a nucleic acid of the invention alone or in combination with one or more other active ingredients in the pharmaceutical compositions of the present invention may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without causing deleterious side effects to the subject or patient. A selected dosage level will depend upon a variety of factors, such as pharmacokinetic factors, including the activity of the particular nucleic acid or composition employed, the route of administration, the time of administration, the rate of excretion of the particular nucleic acid being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the subject or patient being treated, and similar factors well known in the medical arts.

The pharmaceutical composition may be a sterile injectable aqueous suspension or solution, or in a lyophilised form.

The pharmaceutical compositions can be in unit dosage form. In such form, the composition is divided into unit doses containing appropriate quantities of the active component. The unit dosage form can be a packaged preparation, the package containing discrete quantities of the preparations, for example, packeted tablets, capsules, and powders in vials or ampoules. The unit dosage form can also be a capsule, cachet, or tablet itself, or it can be the appropriate number of any of these packaged forms. It may be provided in single dose injectable form, for example in the form of a pen. Compositions may be formulated for any suitable route and means of administration.

The pharmaceutical compositions and medicaments of the present invention may be administered to a mammalian subject in a pharmaceutically effective dose. The mammal may be selected from a human, a non-human primate, a simian or prosimian, a dog, a cat, a horse, cattle, a pig, a goat, a sheep, a mouse, a rat, a hamster, a hedgehog and a guinea pig, or other species of relevance. On this basis, “CNNM4” as used herein denotes nucleic acid or protein in any of the above-mentioned species, if expressed therein naturally or artificially, but preferably this wording denotes human nucleic acids or proteins.

Pharmaceutical compositions of the invention may be administered alone or in combination with one or more other therapeutic or diagnostic agents. A combination therapy may include a nucleic acid of the present invention combined with at least one other therapeutic agent selected based on the particular patient, disease or condition to be treated. Examples of other such agents include, inter alia, a therapeutically active small molecule or polypeptide, a single chain antibody, a classical antibody or fragment thereof, or a nucleic acid molecule which modulates gene expression of one or more additional genes, and similar modulating therapeutics which may complement or otherwise be beneficial in a therapeutic or prophylactic treatment regimen.

Pharmaceutical compositions are typically sterile and stable under the conditions of manufacture and storage. The composition may be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration. The carrier may be a solvent or dispersion medium containing, for example, water, alcohol such as ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), or any suitable mixtures. The proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by use of surfactants according to formulation chemistry well known in the art. In certain embodiments, isotonic agents, e.g., sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride may be desirable in the composition. Prolonged absorption of injectable compositions may be brought about by including in the composition an agent that delays absorption for example, monostearate salts and gelatine.

One aspect of the invention is a nucleic acid or a composition disclosed herein for use as a medicament. The nucleic acid or composition is preferably for use in the prevention, decrease of the risk of suffering from, or treatment of a disease, disorder or syndrome.

The present invention provides a nucleic acid for use, alone or in combination with one or more additional therapeutic agents in a pharmaceutical composition, for treatment or prophylaxis of conditions, diseases and disorders responsive to inhibition of CNNM4 expression.

One aspect of the invention is the use of a nucleic acid or a composition as disclosed herein in the prevention, decrease of the risk of suffering from, or treatment of a disease, disorder or syndrome.

One aspect of the invention is the use of a nucleic acid or a composition as disclosed herein in a method of inhibiting the expression of CNNM4 in a cell, preferably in vitro.

One aspect of the invention is a method of inhibiting the expression of CNNM4 in a cell, preferably in vitro, comprising a step of administering a nucleic acid or a composition as disclosed herein to cells, preferably in vitro.

Nucleic acids and pharmaceutical compositions of the invention may be used in the treatment of a variety of conditions, disorders or diseases. Treatment with a nucleic acid of the invention preferably leads to in vivo CNNM4 depletion, preferably in the liver and/or in blood. As such, nucleic acids of the invention, and compositions comprising them, will be useful in methods for treating a variety of pathological disorders in which inhibiting the expression of CNNM4 may be beneficial. The present invention provides methods for treating a disease, disorder or syndrome comprising the step of administering to a subject in need thereof a therapeutically effective amount of a nucleic acid of the invention.

The invention thus provides methods of treatment or prevention of a disease, disorder or syndrome, the method comprising the step of administering to a subject (e.g., a patient) in need thereof a therapeutically effective amount of a nucleic acid or pharmaceutical composition comprising a nucleic acid of the invention.

The most desirable therapeutically effective amount is an amount that will produce a desired efficacy of a particular treatment selected by one of skill in the art for a given subject in need thereof. This amount will vary depending upon a variety of factors understood by the skilled worker, including but not limited to the characteristics of the therapeutic compound (including activity, pharmacokinetics, pharmacodynamics, and bioavailability), the physiological condition of the subject (including age, sex, disease type and stage, general physical condition, responsiveness to a given dosage, and type of medication), the nature of the pharmaceutically acceptable carrier or carriers in the formulation, and the route of administration. One skilled in the clinical and pharmacological arts will be able to determine a therapeutically effective amount through experimentation, namely by monitoring a subject’s response to administration of a compound and adjusting the dosage accordingly. See, e.g., Remington: The Science and Practice of Pharmacy 21st Ed., Univ. of Sciences in Philadelphia (USIP), Lippincott Williams & Wilkins, Philadelphia, PA, 2005.

In certain embodiments, nucleic acids and pharmaceutical compositions of the invention may be used to treat or prevent a disease, disorder or syndrome.

In certain embodiments, the present invention provides methods for treating a disease, disorder or syndrome in a mammalian subject, such as a human, the method comprising the step of administering to a subject in need thereof a therapeutically effective amount of a nucleic acid as disclosed herein.

Administration of a “therapeutically effective dosage” of a nucleic acid of the invention may result in a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom-free periods, or a prevention of impairment or disability due to the disease affliction.

Nucleic acids of the invention may be beneficial in treating or diagnosing a disease, disorder or syndrome that may be diagnosed or treated using the methods described herein. Treatment and diagnosis of other diseases, disorders or syndromes are also considered to fall within the scope of the present invention.

One aspect of the invention is a method of preventing, decreasing the risk of suffering from, or treating a disease, disorder or syndrome comprising administering a pharmaceutically effective dose or amount a nucleic acid or a composition disclosed herein to an individual in need of treatment, preferably wherein the nucleic acid or composition is administered to the subject subcutaneously, intravenously or by oral, rectal, pulmonary, intramuscular or intraperitoneal administration. Preferably, it is administered subcutaneously.

The disease, disorder or syndrome to be prevented, or treated with a nucleic acid or composition disclosed herein is preferably a disease, disorder or syndrome associated with magnesium dysregulation, preferably hypomagnesemia in the liver.

The disease, disorder or syndrome to be prevented or treated with a nucleic acid or composition disclosed herein is preferably associated with magnesium dysregulation, such as hypomagnesemia in the liver and/or it is associated with aberrant expression and/or overexpression or ectopic expression or localisation or accumulation of CNNM4. The disease, disorder or syndrome to be prevented or treated can have genetic causes or can be acquired.

The disease, disorder or syndrome to be prevented or treated with a nucleic acid or composition disclosed herein is:

-   a) selected from a liver disease, a kidney disease or a lung     disease, preferably a liver disease; -   b) selected from non-alcoholic steatohepatitis (NASH), liver     cirrhosis, hepatocellular carcinoma (HCC), drug-induced liver injury     (DILI), non-alcoholic fatty liver disease (NAFLD), fatty liver,     liver cancer, liver fibrosis, veno-occlusive liver disease, hepatic     sinusoidal obstruction syndrome (SOS), steatosis, Budd-Chiari     syndrome, viral hepatitis B, viral hepatitis C, alcoholic hepatitis,     hepatic ischemia reperfusion injury, primary biliary cirrhosis     (PBC), a chronic liver disease, an acute liver disease, liver     damage, a non-proliferative liver disease, cholangiocarcinoma (bile     duct cancer), a disease associated with hypomagnesemia in the liver,     magnesium dysregulation, kidney fibrosis, acute kidney injury (AKI),     chronic kidney disease, kidney nephritis, kidney nephrosis, lung     cancer, lung adenocarcinoma (LUAD), lung fibrosis, metabolic     syndrome, a cardiovascular disease or complication, obesity, insulin     resistance, diabetes, acetaminophen (APAP)-induced liver injury,     inflammatory response, mitochondrial dysfunction, a decrease of     anti-oxidant system activity, a non-proliferative disease,     arteriosclerosis or any combination thereof; -   c) a liver disease selected from non-alcoholic steatohepatitis     (NASH), liver cirrhosis, hepatocellular carcinoma (HCC),     drug-induced liver injury (DILI), non-alcoholic fatty liver disease     (NAFLD), fatty liver, liver cancer, liver fibrosis, veno-occlusive     liver disease, hepatic sinusoidal obstruction syndrome (SOS),     steatosis, Budd-Chiari syndrome, viral hepatitis B, viral hepatitis     C, alcoholic hepatitis, hepatic ischemia reperfusion injury, primary     biliary cirrhosis (PBC), a chronic liver disease, an acute liver     disease, liver damage, a non-proliferative liver disease     cholangiocarcinoma (bile duct cancer), a disease associated with     hypomagnesemia in the liver, preferably the liver disease is     non-alcoholic steatohepatitis (NASH); -   d) a liver disease selected from non-alcoholic steatohepatitis     (NASH), liver cirrhosis, hepatocellular carcinoma (HCC),     drug-induced liver injury (DILI), non-alcoholic fatty liver disease     (NAFLD) and fatty liver; -   e) a liver disease selected from non-alcoholic steatohepatitis     (NASH), liver cirrhosis, liver fibrosis, veno-occlusive liver     disease, drug-induced liver injury (DILI), hepatocellular carcinoma     (HCC) and Budd-Chiari syndrome and hepatitis; -   f) a liver disease selected from non-alcoholic steatohepatitis     (NASH), liver cirrhosis, liver fibrosis, drug-induced liver injury     (DILI) and hepatocellular carcinoma (HCC); -   g) a liver disease selected from non-alcoholic steatohepatitis     (NASH), drug-induced liver injury (DILI) and liver cirrhosis; -   h) non-alcoholic fatty liver disease (NAFLD); -   i) non-alcoholic steatohepatitis (NASH); -   j) drug-induced liver injury (DILI); -   k) a kidney disease, preferably selected from kidney fibrosis, acute     kidney injury (AKI), a chronic kidney disease, kidney nephritis and     kidney nephrosis, and most preferably the kidney disease is kidney     fibrosis; and/or -   l) a lung disease, preferably selected from lung cancer, lung     adenocarcinoma (LUAD) and liver fibrosis, and most preferably the     lung disease is lung adenocarcinoma (LUAD).

Liver cancer is preferably hepatocellular carcinoma (HCC) or cholangiocarcinoma (bile duct cancer).

In one embodiment, nucleic acids or compositions of the invention are for use or are used in a method of treatment to:

-   a) increase the Mg²⁺ level in the liver of a subject; -   b) reduce the Mg²⁺ level in the plasma or serum of a subject; -   c) restore Mg²⁺ homeostasis in the liver of a subject; -   d) reduce the lipid content in the liver of a subject; -   e) reduce inflammation by reactive oxygen species (ROS), preferably     in the liver of a subject; -   f) reduce the reactive oxygen species (ROS) level, preferably in the     liver of a subject; -   g) reduce inflammation, preferably in the liver of a subject; and/or -   h) reduce fibrosis, preferably in the liver of a subject.

Preferably, the use of a nucleic acid or composition disclosed herein increases the Mg²⁺ level in the liver of a subject treated with the nucleic acid or composition to the corresponding level expected in a healthy subject. Alternatively, it increases the Mg²⁺ level in the liver of the subject treated with the nucleic acid or composition such that the difference between the Mg²⁺ level in the liver of the subject before treatment and the corresponding level expected in a healthy subject is at least temporarily reduced by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%.

Preferably, the use of a nucleic acid or composition disclosed herein reduces the Mg²⁺ level in the plasma or serum of a subject treated with the nucleic acid or composition to the corresponding level expected in a healthy subject. Alternatively, it reduces the Mg²⁺ level in the plasma or serum of the subject treated with the nucleic acid or composition such that the difference between the Mg²⁺ level in the plasma or serum of the subject before treatment and the corresponding level expected in a healthy subject is at least temporarily reduced by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%.

Preferably, the use of a nucleic acid or composition disclosed herein reduces the lipid content in the liver of a subject treated with the nucleic acid or composition to the corresponding level expected in a healthy subject. Alternatively, it reduces the lipid content in the liver of the subject treated with the nucleic acid or composition such that the difference between the lipid content in the liver of the subject before treatment and the corresponding level expected in a healthy subject is at least temporarily reduced by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%.

Preferably, the use of a nucleic acid or composition disclosed herein reduces inflammation by reactive oxygen species (ROS) in the liver of a subject treated with the nucleic acid or composition to the corresponding level expected in a healthy subject. Alternatively, it reduces the inflammation by reactive oxygen species (ROS) in the liver of the subject treated with the nucleic acid or composition such that the difference between the inflammation by reactive oxygen species (ROS) in the liver of the subject before treatment and the corresponding level expected in a healthy subject is at least temporarily reduced by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%.

Preferably, the use of a nucleic acid or composition disclosed herein reduces reactive oxygen species (ROS) level in the liver of a subject treated with the nucleic acid or composition to the corresponding level expected in a healthy subject. Alternatively, it reduces the reactive oxygen species (ROS) level in the liver of a subject treated with the nucleic acid or composition to a level such that the difference between the reactive oxygen species (ROS) level in the liver of the subject before treatment and the corresponding value expected in a healthy subject is at least temporarily reduced by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%.

Preferably, the use of a nucleic acid or composition disclosed herein reduces inflammation in the liver of a subject treated with the nucleic acid or composition to the corresponding level expected in a healthy subject. Alternatively, it reduces the inflammation in the liver of the subject treated with the nucleic acid or composition such that the difference between the inflammation in the liver of the subject before treatment and the corresponding level expected in a healthy subject is at least temporarily reduced by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%.

Preferably, the use of a nucleic acid or composition disclosed herein reduces fibrosis in the liver of a subject treated with the nucleic acid or composition to the corresponding level expected in a healthy subject. Alternatively, it reduces the fibrosis in the liver of the subject treated with the nucleic acid or composition such that the difference between the fibrosis in the liver of the subject before treatment and the corresponding level expected in a healthy subject is at least temporarily reduced by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%. Fibrosis in the liver of a subject can be measured for example by measuring the level of alpha smooth muscle actin (αSMA) or collagen deposition in the liver of the subject.

It is evident that an appropriate dosage regimen of a nucleic acid or composition is necessary to achieve these outcomes. The skilled person will be able to determine the dosage regimen necessary to achieve these outcomes.

Liver diseases in general, but particularly liver diseases caused by drugs (drug-induced liver injury or DILI), manifest themselves clinically with a variety of symptoms which as such are not particular informative. Non-limiting examples of symptoms include: loss of appetite, exhaustion, giddiness, weight loss, nausea, vomiting, fever, pain in the upper right abdominal region, arthralgias, myalgias, itching, rashes, discoloration of excretions may be mentioned, yellowing of the eyes and of the skin.

As an expert in the field knows, the presence of active liver disease is often detected by the existence of elevated enzyme levels in the blood. Specifically, blood levels of ALT (alanine aminotransferase) and AST (aspartate aminotransferase) above clinically accepted normal ranges are known to be indicative of on-going liver damage. Routine monitoring of liver disease patients for blood levels of ALT and AST is used clinically to measure progress of the liver disease while on medical treatment. Reduction of elevated ALT and AST to within the accepted normal range is taken as clinical evidence reflecting a reduction in the severity of the patient on-going liver damage. In a particular embodiment, the liver disease is caused by any kind of liver damage.

A nucleic acid or compositions disclosed herein may be for use in a regimen comprising treatments once or twice weekly, every week, every two weeks, every three weeks, every four weeks, every five weeks, every six weeks, every seven weeks, every eight weeks, every nine weeks, every ten weeks, every eleven weeks, every twelve weeks, every three months, every four months, every five months, every six months or in regimens with varying dosing frequency such as combinations of the before-mentioned intervals. The nucleic acid or composition may be for use subcutaneously, intravenously or using any other application routes such as oral, rectal, pulmonary, or intraperitoneal. Preferably, it is for use subcutaneously.

An exemplary treatment regime is administration once every two weeks, once every three weeks, once every four weeks, once a month, once every two or three months or once every four, five or six or more months. Dosages may be selected and readjusted by the skilled health care professional as required to maximize therapeutic benefit for a particular subject, e.g., patient. The nucleic acids will typically be administered on multiple occasions. Intervals between single dosages can be, for example, 2-5 days, weekly, bi-weekly, monthly, every two or three months, every four or five months, every six months, or yearly. Intervals between administrations can also be irregular, based on nucleic acid target gene product levels for example in the blood or liver of the subject or patient.

In cells and/or subjects treated with or receiving a nucleic acid or composition as disclosed herein, the CNNM4 expression may be inhibited compared to untreated cells and/or subjects by a range from 15% up to 100% but at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 100% or intermediate values. The level of inhibition may allow treatment of a disease associated with CNNM4 expression or overexpression or hypomagnesemia, preferably in the liver, or may serve to further investigate the functions and physiological roles of CNNM4 gene products. The level of inhibition is preferably measured in the liver or in the blood or in the kidneys, preferably in the liver, of the subject treated with the nucleic acid or composition.

One aspect is the use of a nucleic acid or composition as disclosed herein in the manufacture of a medicament for treating a disease, disorder or syndromes, such as those as listed above or additional pathologies associated with elevated levels of CNNM4, preferably in the liver or in the kidneys, or hypomagnesemia, preferably in the liver, or additional therapeutic approaches where inhibition of CNNM4 expression is desired. A medicament is a pharmaceutical composition.

Each of the nucleic acids of the invention and pharmaceutically acceptable salts and solvates thereof constitutes an individual embodiment of the invention.

Also included in the invention is a method of treating or preventing a disease, disorder or syndrome, such as those listed above, comprising administration of a composition comprising a nucleic acid or composition as described herein, to an individual in need of treatment (to improve such pathologies). The nucleic acid or composition may be administered in a regimen comprising treatments twice every week, once every week, every two weeks, every three weeks, every four weeks, every five weeks, every six weeks, every seven weeks, or every eight to twelve or more weeks or in regimens with varying dosing frequency such as combinations of the before-mentioned intervals. The nucleic acid or conjugated nucleic acid may be for use subcutaneously or intravenously or other application routes such as oral, rectal or intraperitoneal.

A nucleic acid of the invention may be administered by any appropriate administration pathway known in the art, including but not limited to aerosol, enteral, nasal, ophthalmic, oral, parenteral, rectal, vaginal, or transdermal (e.g., topical administration of a cream, gel or ointment, or by means of a transdermal patch). “Parenteral administration” is typically associated with injection at or in communication with the intended site of action, including infraorbital, infusion, intraarterial, intracapsular, intracardiac, intradermal, intramuscular, intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrathecal, intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous, transmucosal, or transtracheal administration.

The use of a chemical modification pattern of the nucleic acids confers nuclease stability in serum and makes for example subcutaneous application route feasible.

Solutions or suspensions used for intradermal or subcutaneous application typically include one or more of: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates; and/or tonicity adjusting agents such as, e.g., sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide, or buffers with citrate, phosphate, acetate and the like. Such preparations may be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

Sterile injectable solutions may be prepared by incorporating a nucleic acid in the required amount in an appropriate solvent with one or a combination of ingredients described above, as required, followed by sterilization microfiltration. Dispersions may be prepared by incorporating the active compound into a sterile vehicle that contains a dispersion medium and optionally other ingredients, such as those described above. In the case of sterile powders for the preparation of sterile injectable solutions, the methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient in addition to any additional desired ingredient from a sterile-filtered solution thereof.

When a therapeutically effective amount of a nucleic acid of the invention is administered by, e.g., intravenous, cutaneous or subcutaneous injection, the nucleic acid will be in the form of a pyrogen-free, parenterally acceptable aqueous solution. Methods for preparing parenterally acceptable solutions, taking into consideration appropriate pH, isotonicity, stability, and the like, are within the skill in the art. A preferred pharmaceutical composition for intravenous, cutaneous, or subcutaneous injection will contain, in addition to a nucleic acid, an isotonic vehicle such as sodium chloride injection, Ringer’s injection, dextrose injection, dextrose and sodium chloride injection, lactated Ringer’s injection, or other vehicle as known in the art. A pharmaceutical composition of the present invention may also contain stabilizers, preservatives, buffers, antioxidants, or other additives well known to those of skill in the art.

The amount of nucleic acid which can be combined with a carrier material to produce a single dosage form will vary depending on a variety of factors, including the subject being treated, and the particular mode of administration. In general, it will be an amount of the composition that produces an appropriate therapeutic effect under the particular circumstances. Generally, out of one hundred percent, this amount will range from about 0.01% to about 99% of nucleic acid, from about 0.1% to about 70%, or from about 1% to about 30% of nucleic acid in combination with a pharmaceutically acceptable carrier.

The nucleic acid may be prepared with carriers that will protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are patented or generally known to those skilled in the art. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

Dosage regimens may be adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a dose may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the particular circumstances of the therapeutic situation, on a case by case basis. It is especially advantageous to formulate parenteral compositions in dosage unit forms for ease of administration and uniformity of dosage when administered to the subject or patient. As used herein, a dosage unit form refers to physically discrete units suitable as unitary dosages for the subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce a desired therapeutic effect. The specification for the dosage unit forms of the invention depend on the specific characteristics of the active compound and the particular therapeutic effect(s) to be achieved and the treatment and sensitivity of any individual patient.

The nucleic acid or composition of the present invention can be produced using routine methods in the art including chemical synthesis, such as solid phase chemical synthesis.

Nucleic acids or compositions of the invention may be administered with one or more of a variety of medical devices known in the art. For example, in one embodiment, a nucleic acid of the invention may be administered with a needleless hypodermic injection device. Examples of well-known implants and modules useful in the present invention are in the art, including e.g., implantable micro-infusion pumps for controlled rate delivery; devices for administering through the skin; infusion pumps for delivery at a precise infusion rate; variable flow implantable infusion devices for continuous drug delivery; and osmotic drug delivery systems. These and other such implants, delivery systems, and modules are known to those skilled in the art.

In certain embodiments, the nucleic acid or composition of the invention may be formulated to ensure a desired distribution in vivo. To target a therapeutic compound or composition of the invention to a particular in vivo location, they can be formulated, for example, in liposomes which may comprise one or more moieties that are selectively transported into specific cells or organs, thus enhancing targeted drug delivery.

The invention is characterized by high specificity at the molecular and tissue-directed delivery level. The sequences of the nucleic acids of the invention are highly specific for their target, meaning that they do not inhibit the expression of genes that they are not designed to target or only minimally inhibit the expression of genes that they are not designed to target and/or only inhibit the expression of a low number of genes that they are not designed to target. A further level of specificity is achieved when nucleic acids are linked to a ligand that is specifically recognised and internalised by a particular cell type. This is for example the case when a nucleic acid is linked to a ligand comprising GalNAc moieties, which are specifically recognised and internalised by hepatocytes. This leads to the nucleic acid inhibiting the expression of their target only in the cells that are targeted by the ligand to which they are linked. These two levels of specificity potentially confer a better safety profile than the currently available treatments. In certain embodiments, the present invention thus provides nucleic acids of the invention linked to a ligand comprising one or more GalNAc moieties, or comprising one or more other moieties that confer cell-type or tissue-specific internalisation of the nucleic acid thereby conferring additional specificity of target gene knockdown by RNA interference.

The nucleic acid as described herein may be formulated with a lipid in the form of a liposome. Such a formulation may be described in the art as a lipoplex. The composition with a lipid/liposome may be used to assist with delivery of the nucleic acid of the invention to the target cells. The lipid delivery system herein described may be used as an alternative to a conjugated ligand. The modifications herein described may be present when using the nucleic acid of the invention with a lipid delivery system or with a ligand conjugate delivery system.

Such a lipoplex may comprise a lipid composition comprising:

-   i) a cationic lipid, or a pharmaceutically acceptable salt thereof; -   ii) a steroid; -   iii) a phosphatidylethanolamine phospholipid; and/or -   iv) a PEGylated lipid.

The cationic lipid may be an amino cationic lipid.

The content of the cationic lipid component may be from about 55 mol% to about 65 mol% of the overall lipid content of the composition. In particular, the cationic lipid component is about 59 mol% of the overall lipid content of the composition.

The compositions can further comprise a steroid. The steroid may be cholesterol. The content of the steroid may be from about 26 mol% to about 35 mol% of the overall lipid content of the lipid composition. More particularly, the content of steroid may be about 30 mol% of the overall lipid content of the lipid composition.

The phosphatidylethanolamine phospholipid may be selected from the group consisting of 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (DPhyPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE), 1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-Dilinoleoyl-sn-glycero-3-phosphoethanolamine (DLoPE), 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1,2-Dierucoyl-sn-glycero-3-phosphoethanolamine (DEPE), 1,2-Disqualeoyl-sn-glycero-3-phosphoethanolamine (DSQPE) and 1-Stearoyl-2-linoleoyl-sn-glycero-3-phosphoethanolamine (SLPE). The content of the phospholipid may be about 10 mol% of the overall lipid content of the composition.

The PEGylated lipid may be selected from the group consisting of 1,2-dimyristoyl-sn-glycerol, methoxypolyethylene glycol (DMG-PEG) and C16-Ceramide-PEG. The content of the PEGylated lipid may be about 1 to 5 mol% of the overall lipid content of the composition.

The content of the cationic lipid component in the composition may be from about 55 mol% to about 65 mol% of the overall lipid content of the lipid composition, preferably about 59 mol% of the overall lipid content of the lipid composition.

The composition may have a molar ratio of the components of i):ii):iii):iv) selected from 55:34:10:1; 56:33:10:1; 57:32:10:1; 58:31:10:1; 59:30:10:1; 60:29:10:1; 61:28:10:1; 62:27:10:1; 63:26:10:1; 64:25:10:1; and 65:24:10:1.

Neutral liposome compositions may be formed from, for example, dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions may be formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes may be formed primarily from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposomal composition may be formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC. Another type is formed from mixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

A positively charged synthetic cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) can be used to form small liposomes that interact spontaneously with nucleic acid to form lipid-nucleic acid complexes which are capable of fusing with the negatively charged lipids of the cell membranes of tissue culture cells. DOTMA analogues can also be used to form liposomes.

Derivatives and analogues of lipids described herein may also be used to form liposomes.

A liposome containing a nucleic acid can be prepared by a variety of methods. In one example, the lipid component of a liposome is dissolved in a detergent so that micelles are formed with the lipid component. For example, the lipid component can be an amphipathic cationic lipid or lipid conjugate. The detergent can have a high critical micelle concentration and may be nonionic. Exemplary detergents include cholate, CHAPS, octylglucoside, deoxycholate, and lauroyl sarcosine. The nucleic acid preparation is then added to the micelles that include the lipid component. The cationic groups on the lipid interact with the nucleic acid and condense around the nucleic acid to form a liposome. After condensation, the detergent is removed, e.g., by dialysis, to yield a liposomal preparation of nucleic acid.

If necessary, a carrier compound that assists in condensation can be added during the condensation reaction, e.g., by controlled addition. For example, the carrier compound can be a polymer other than a nucleic acid (e.g., spermine or spermidine). pH can also be adjusted to favour condensation.

Nucleic acid formulations of the present invention may include a surfactant. In one embodiment, the nucleic acid is formulated as an emulsion that includes a surfactant.

A surfactant that is not ionized is a non-ionic surfactant. Examples include non-ionic esters, such as ethylene glycol esters, propylene glycol esters, glyceryl esters etc., nonionic alkanolamides, and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylated block polymers.

A surfactant that carries a negative charge when dissolved or dispersed in water is an anionic surfactant. Examples include carboxylates, such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates.

A surfactant that carries a positive charge when dissolved or dispersed in water is a cationic surfactant. Examples include quaternary ammonium salts and ethoxylated amines.

A surfactant that has the ability to carry either a positive or negative charge is an amphoteric surfactant. Examples include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines and phosphatides.

“Micelles” are defined herein as a particular type of molecular assembly in which amphipathic molecules are arranged in a spherical structure such that all the hydrophobic portions of the molecules are directed inward, leaving the hydrophilic portions in contact with the surrounding aqueous phase. The converse arrangement exists if the environment is hydrophobic. A micelle may be formed by mixing an aqueous solution of the nucleic acid, an alkali metal alkyl sulphate, and at least one micelle forming compound.

Exemplary micelle forming compounds include lecithin, hyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid, glycolic acid, lactic acid, chamomile extract, cucumber extract, oleic acid, linoleic acid, linolenic acid, monoolein, monooleates, monolaurates, borage oil, evening of primrose oil, menthol, trihydroxy oxo cholanyl glycine and pharmaceutically acceptable salts thereof, glycerol, polyglycerol, lysine, polylysine, triolein, polyoxyethylene ethers and analogues thereof, polidocanol alkyl ethers and analogues thereof, chenodeoxycholate, deoxycholate, and mixtures thereof.

Phenol and/or m-cresol may be added to the mixed micellar composition to act as a stabiliser and preservative. An isotonic agent such as glycerine may as be added.

A nucleic acid preparation may be incorporated into a particle such as a microparticle. Microparticles can be produced by spray-drying, lyophilisation, evaporation, fluid bed drying, vacuum drying, or a combination of these methods.

Definitions

As used herein, the terms “inhibit”, “down-regulate”, or “reduce” with respect to gene expression mean that the expression of the gene, or the level of RNA molecules or equivalent RNA molecules encoding one or more proteins or protein subunits (e.g., mRNA), or the activity of one or more proteins or protein subunits, is reduced below that observed either in the absence of the nucleic acid or conjugated nucleic acid of the invention or as compared to that obtained with an siRNA molecule with no known homology to the human transcript (herein termed non-silencing control). Such control may be conjugated and modified in an analogous manner to the molecule of the invention and delivered into the target cell by the same route. The expression after treatment with the nucleic acid of the invention may be reduced to 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 15%, 10%, 5% or 0% or to intermediate values, or less than that observed in the absence of the nucleic acid or conjugated nucleic acid. The expression may be measured in the cells to which the nucleic acid is applied. Alternatively, especially if the nucleic acid is administered to a subject, the level can be measured in a different group of cells or in a tissue or an organ or in a body fluid such as blood or plasma. The level of inhibition is preferably measured in conditions that have been selected because they show the greatest effect of the nucleic acid on the target mRNA level in cells treated with the nucleic acid in vitro. The level of inhibition may for example be measured after 24 hours or 48 hours of treatment with a nucleic acid at a concentration of between 0.038 nM- 10 µM, preferably 1 nM, 10 nM or 100 nM. These conditions may be different for different nucleic acid sequences or for different types of nucleic acids, such as for nucleic acids that are unmodified or modified or conjugated to a ligand or not. Examples of suitable conditions for determining levels of inhibition are described in the examples.

By nucleic acid it is meant a nucleic acid comprising two strands comprising nucleotides, that is able to interfere with gene expression. Inhibition may be complete or partial and results in down regulation of gene expression in a targeted manner. The nucleic acid comprises two separate polynucleotide strands; the first strand, which may also be a guide strand; and a second strand, which may also be a passenger strand. The first strand and the second strand may be part of the same polynucleotide molecule that is self-complementary which ‘folds’ back to form a double-stranded molecule. The nucleic acid may be an siRNA molecule.

The nucleic acid may comprise ribonucleotides, modified ribonucleotides, deoxynucleotides, deoxyribonucleotides, or nucleotide analogues non-nucleotides that are able to mimic nucleotides such that they may ‘pair’ with the corresponding base on the target sequence or complementary strand. The nucleic acid may further comprise a double-stranded nucleic acid portion or duplex region formed by all or a portion of the first strand (also known in the art as a guide strand) and all or a portion of the second strand (also known in the art as a passenger strand). The duplex region is defined as beginning with the first base pair formed between the first strand and the second strand and ending with the last base pair formed between the first strand and the second strand, inclusive.

By duplex region it is meant the region in two complementary or substantially complementary oligonucleotides that form base pairs with one another, either by Watson-Crick base pairing or any other manner that allows for a duplex between oligonucleotide strands that are complementary or substantially complementary. For example, an oligonucleotide strand having 21 nucleotide units can base pair with another oligonucleotide of 21 nucleotide units, yet only 19 nucleotides on each strand are complementary or substantially complementary, such that the “duplex region” consists of 19 base pairs. The remaining base pairs may exist as 5′ and 3′ overhangs, or as single-stranded regions. Further, within the duplex region, 100% complementarity is not required; substantial complementarity is allowable within a duplex region. Substantial complementarity refers to complementarity between the strands such that they are capable of annealing under biological conditions. Techniques to empirically determine if two strands are capable of annealing under biological conditions are well known in the art. Alternatively, two strands can be synthesised and added together under biological conditions to determine if they anneal to one another. The portion of the first strand and second strand that forms at least one duplex region may be fully complementary and is at least partially complementary to each other. Depending on the length of a nucleic acid, a perfect match in terms of base complementarity between the first strand and the second strand is not necessarily required. However, the first and second strands must be able to hybridise under physiological conditions.

As used herein, the terms “non-pairing nucleotide analogue” means a nucleotide analogue which includes a non-base pairing moiety including but not limited to: 6 des amino adenosine (Nebularine), 4-Me-indole, 3-nitropyrrole, 5-nitroindole, Ds, Pa, N3-Me ribo U, N3-Me riboT, N3-Me dC, N3-Me-dT, N1-Me-dG, N1-Me-dA, N3-ethyl-dC, and N3-Me dC. In some embodiments the non-base pairing nucleotide analogue is a ribonucleotide. In other embodiments it is a deoxyribonucleotide.

As used herein, the term, “terminal functional group” includes without limitation a halogen, alcohol, amine, carboxylic, ester, amide, aldehyde, ketone, and ether groups.

An “overhang” as used herein has its normal and customary meaning in the art, i.e. a single-stranded portion of a nucleic acid that extends beyond the terminal nucleotide of a complementary strand in a double-strand nucleic acid. The term “blunt end” includes double-stranded nucleic acid whereby both strands terminate at the same position, regardless of whether the terminal nucleotide(s) are base-paired. The terminal nucleotide of a first strand and a second strand at a blunt end may be base paired. The terminal nucleotide of a first strand and a second strand at a blunt end may not be paired. The terminal two nucleotides of a first strand and a second strand at a blunt end may be base-paired. The terminal two nucleotides of a first strand and a second strand at a blunt end may not be paired.

The term “serinol-derived linker moiety” means the linker moiety comprises the following structure:

An O atom of said structure typically links to an RNA strand and the N atom typically links to the targeting ligand.

The terms “patient,” “subject,” and “individual” may be used interchangeably and refer to either a human or a non-human animal. These terms include mammals such as humans, primates, livestock animals (e.g., bovines, porcines), companion animals (e.g., canines, felines) and rodents (e.g., mice and rats).

As used herein, “treating” or “treatment” and grammatical variants thereof refer to an approach for obtaining beneficial or desired clinical results. The term may refer to slowing the onset or rate of development of a condition, disorder or disease, reducing or alleviating symptoms associated with it, generating a complete or partial regression of the condition, or some combination of any of the above. For the purposes of this invention, beneficial or desired clinical results include, but are not limited to, reduction or alleviation of symptoms, diminishment of extent of disease, stabilization (i.e., not worsening) of state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. “Treatment” can also mean prolonging survival relative to expected survival time if not receiving treatment. A subject (e.g., a human) in need of treatment may thus be a subject already afflicted with the disease or disorder in question. The term “treatment” includes inhibition or reduction of an increase in severity of a pathological state or symptoms relative to the absence of treatment, and is not necessarily meant to imply complete cessation of the relevant disease, disorder or condition.

As used herein, the terms “preventing” and grammatical variants thereof refer to an approach for preventing the development of, or altering the pathology of, a condition, disease or disorder. Accordingly, “prevention” may refer to prophylactic or preventive measures. For the purposes of this invention, beneficial or desired clinical results include, but are not limited to, prevention or slowing of symptoms, progression or development of a disease, whether detectable or undetectable. A subject (e.g., a human) in need of prevention may thus be a subject not yet afflicted with the disease or disorder in question. The term “prevention” includes slowing the onset of disease relative to the absence of treatment, and is not necessarily meant to imply permanent prevention of the relevant disease, disorder or condition. Thus “preventing” or “prevention” of a condition may in certain contexts refer to reducing the risk of developing the condition, or preventing or delaying the development of symptoms associated with the condition.

As used herein, an “effective amount,” “therapeutically effective amount” or “effective dose” is an amount of a composition (e.g., a therapeutic composition or agent) that produces at least one desired therapeutic effect in a subject, such as preventing or treating a target condition or beneficially alleviating a symptom associated with the condition.

As used herein, the term “pharmaceutically acceptable salt” refers to a salt that is not harmful to a patient or subject to which the salt in question is administered. It may be a salt chosen, e.g., among acid addition salts and basic salts. Examples of acid addition salts include chloride salts, citrate salts and acetate salts. Examples of basic salts include salts wherein the cation is selected from alkali metal cations, such as sodium or potassium ions, alkaline earth metal cations, such as calcium or magnesium ions, as well as substituted ammonium ions, such as ions of the type N(R¹)(R²)(R³)(R⁴)⁺, wherein R¹, R², R³ and R⁴ independently will typically designate hydrogen, optionally substituted C1-6-alkyl groups or optionally substituted C2-6-alkenyl groups. Examples of relevant C1-6-alkyl groups include methyl, ethyl, 1-propyl and 2-propyl groups. Examples of C2-6-alkenyl groups of possible relevance include ethenyl, 1-propenyl and 2-propenyl. Other examples of pharmaceutically acceptable salts are described in “Remington’s Pharmaceutical Sciences”, 17th edition, Alfonso R. Gennaro (Ed.), Mark Publishing Company, Easton, PA, USA, 1985 (and more recent editions thereof), in the “Encyclopaedia of Pharmaceutical Technology”, 3rd edition, James Swarbrick (Ed.), Informa Healthcare USA (Inc.), NY, USA, 2007, and in J. Pharm. Sci. 66: 2 (1977). A “pharmaceutically acceptable salt” retains qualitatively a desired biological activity of the parent compound without imparting any undesired effects relative to the compound. Examples of pharmaceutically acceptable salts include acid addition salts and base addition salts. Acid addition salts include salts derived from nontoxic inorganic acids, such as hydrochloric, nitric, phosphorous, phosphoric, sulfuric, hydrobromic, hydroiodic and the like, or from nontoxic organic acids such as aliphatic mono- and di-carboxylic acids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids and the like. Base addition salts include salts derived from alkaline earth metals, such as sodium, potassium, magnesium, calcium and the like, as well as from nontoxic organic amines, such as N, N′-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, procaine and the like.

The term “pharmaceutically acceptable carrier” includes any of the standard pharmaceutical carriers. Pharmaceutically acceptable carriers for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington’s Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985). For example, sterile saline and phosphate- buffered saline at slightly acidic or physiological pH may be used. Exemplary pH buffering agents include phosphate, citrate, acetate, tris/hydroxymethyl)aminomethane (TRIS), N-Tris(hydroxymethyl)methyl-3-aminopropanesulphonic acid (TAPS), ammonium bicarbonate, diethanolamine, histidine, which is a preferred buffer, arginine, lysine, or acetate or mixtures thereof. The term further encompasses any agents listed in the US Pharmacopeia for use in animals, including humans. A “pharmaceutically acceptable carrier” includes any and all physiologically acceptable, i.e., compatible, solvents, dispersion media, coatings, antimicrobial agents, isotonic and absorption delaying agents, and the like. In certain embodiments, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). Depending on selected route of administration, the nucleic acid may be coated in a material or materials intended to protect the compound from the action of acids and other natural inactivating conditions to which the nucleic acid may be exposed when administered to a subject by a particular route of administration.

The term “solvate” in the context of the present invention refers to a complex of defined stoichiometry formed between a solute (in casu, a nucleic acid compound or pharmaceutically acceptable salt thereof according to the invention) and a solvent. The solvent in this connection may, for example, be water or another pharmaceutically acceptable, typically small-molecular organic species, such as, but not limited to, acetic acid or lactic acid. When the solvent in question is water, such a solvate is normally referred to as a hydrate.

As it is used herein, the term “CNNM4” means “Cyclin and CBS domain divalent metal cation transport mediator” (also known as Ancient Conserved Domain Protein, ACDP, Cyclin M or CNNM). CNNMs are membrane proteins encoded by four genes, CNNM1, CNNM2, CNNM3 and CNNM4 that are evolutionary expressed throughout development and in all adult tissues, except for CNNM1 which is mainly expressed in the brain. CNNMs play a key role in the transport of magnesium ions (Mg²⁺) through the cell membranes in different organs (Funato et al., 2014. J Clin Invest. 124(12):5398-5410). CNNM4 corresponds to the human gene identified by ID number ENSG00000158158 in the Ensembl database (according to release 93 of July 2018). According to the Ensembl database, CNNM4 encodes at least 4 transcript or splice variants. Thus, the disclosure relates to variant CNNM4-201 (ENST00000377075.2) and to the other three variants CNNM4-204 (ENST00000496186.5), CNNM4-203 (ENST00000493384.1) and CNNM4-202 (ENST00000482716.5). CNNM4 gene encodes the protein “Metal transporter CNNM4” identified by the Uniprot Database as Q6P4Q7 (according to version 130 of Oct. 10, 2018).

As used herein, a “liver disease” is an acute or chronic damage to the liver, usually caused by infection, injury, exposure to drugs or toxic compounds, alcohol, impurities in foods, and the abnormal build-up of normal substances in the blood, an autoimmune process, or by a genetic defect (such as haemochromatosis). Sometimes the exact cause of the disease or injury may not be known. Liver disease can be classified as acute or chronic liver disease based in the duration of the disease. In acute liver diseases, such as acute hepatitis and acute liver failure (ALF), the history of the disease does not exceed six months. Liver diseases of longer duration are classified as chronic liver diseases. Non-limiting examples of common liver diseases include cirrhosis, liver fibrosis, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), hepatic ischemia reperfusion injury, primary biliary cirrhosis (PBC), and hepatitis, including viral and alcoholic hepatitis. Most common forms of viral hepatitis are hepatitis B and C (HBV and HCV, respectively). Chronic hepatitis may result in cirrhosis. The death of liver cells through a process known as apoptosis is common in all forms of liver diseases. Apoptosis of liver cells is linked to liver fibrosis and other liver disease.

The term “liver damage”, as used herein, is used to denote any type of hepatic trauma (injury), including chronic and acute trauma as well as pathological change present in liver cell or tissue. The clinical conditions of liver damage may include, without being limited thereto, degeneration of live cells, vasculitis of liver, spotty necrosis or focal necrosis present in liver, inflammatory cell infiltration or fibroblast proliferation in liver and portal area, or hepatomegaly, and hepatocirrhosis, hepatoma resulted from severe liver damage, and the like. Liver disease results from an injury to the liver. The injury to the liver may be caused by toxins, including alcohol, some drugs, impurities in foods, the abnormal build-up of normal substances in the blood, by infection or by an autoimmune disorder. In some cases, the liver damage resulting from an injury to the liver include, but is not limited to fatty liver, cirrhosis, primary biliary cirrhosis, primary sclerosing cholangitis, and alpha1- antitrypsin deficiency. The liver damage includes, but is not limited to cirrhosis, liver fibrosis, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), hepatic ischemia reperfusion injury, hepatitis, including viral and alcoholic hepatitis and primary biliary cirrhosis (PBC).

As used herein, “fibrosis” is the formation of excess fibrous connective tissue in an organ or tissue in a reparative or reactive process. This can be a reactive, benign, or pathological state. In response to injury, this is called scarring, and if fibrosis arises from a single cell line, this is called a fibroma. Physiologically, fibrosis acts to deposit connective tissue, which can interfere with or totally inhibit the normal architecture and function of the underlying organ or tissue. Fibrosis can be used to describe the pathological state of excess deposition of fibrous tissue, as well as the process of connective tissue deposition in healing. Fibrosis is defined by the pathological accumulation of extracellular matrix (ECM) proteins and results in scarring and thickening of the affected tissue. It is in essence an exaggerated wound healing response which interferes with normal organ function. Fibrosis may affect any organ. In a preferred embodiment, fibrosis is liver fibrosis.

As used herein, “liver fibrosis” is the scarring process that represents the liver’s response to injury. In the same way as skin and other organs heal wounds through deposition of collagen and other matrix constituents so the liver repairs injury through the deposition of new collagen. Liver fibrosis is the first stage of liver scarring. Later, if more of the liver becomes scarred, it may lead to liver cirrhosis.

As used herein, “veno-occlusive liver disease” or “hepatic sinusoidal obstruction syndrome (SOS)” is characterized by hepatomegaly, right upper quadrant pain, jaundice, and ascites, most often occurring in patients undergoing hematopoietic cell transplantation (HCT) and less commonly following the use of certain chemotherapeutic agents in non-transplant settings, ingestion of alkaloid toxins, after high dose radiation therapy, or liver transplantation. The hepatic venous outflow obstruction in SOS is due to occlusion of the terminal hepatic venules and hepatic sinusoids

As used herein, “drug-induced liver injury (DILI)” means injury to the liver developed following the use of one or more drugs.

As used herein, “steatosis”, also called fatty change, is the process describing the abnormal retention of lipids within a cell. It reflects an impairment of the normal processes of synthesis and elimination of triglyceride fat. Excess lipid accumulates in vesicles that displace the cytoplasm. When the vesicles are large enough to distort the nucleus, the condition is known as macrovesicular steatosis; otherwise, the condition is known as microvesicular steatosis. While not particularly detrimental to the cell in mild cases, large accumulations can disrupt cell constituents, and in severe cases the cell may even burst. The risk factors associated with steatosis are varied, and include diabetes mellitus, protein malnutrition, hypertension, cell toxins, obesity, anoxia, and sleep apnoea. As the liver is the primary organ of lipid metabolism it is most often associated with steatosis; however, it may occur in any organ, commonly the kidneys, heart, and muscle. In a preferred embodiment, the steatosis is caused in the liver.

As used herein, “non-alcoholic steatohepatitis (NASH)” is a liver syndrome. It causes liver damage that is histologically indistinguishable from alcoholic hepatitis. It can however also be present patients who do not suffer from alcohol dependence. NASH develops most often in patients with at least one of the following risk factors: obesity, dyslipidaemia, and glucose intolerance. The pathogenesis is not always well understood but seems to be linked to insulin resistance (e.g., as in obesity or metabolic syndrome). Most patients are asymptomatic. Laboratory findings include elevation in aminotransferase levels. Biopsy is required to confirm the diagnosis.

As used herein, “cirrhosis”, means a late stage of progressive hepatic fibrosis characterized by distortion of the hepatic architecture and the formation of regenerative nodules. It is generally considered to be irreversible in its advanced stages, at which point the only treatment option may be liver transplantation. Reversal of cirrhosis (in its earlier stages) is possible in several forms of liver disease following treatment of the underlying cause. Patients with cirrhosis are susceptible to a variety of complications, and their life expectancy is usually short.

As used herein, “hepatocellular carcinoma (HCC)”, means an aggressive tumour that often occurs in the setting of chronic liver disease and cirrhosis. It is typically diagnosed late in its course, and the median survival following diagnosis is approximately 6 to 20 months.

Although the mainstay of therapy is surgical resection, the majority of patients are not eligible because of tumour extent or underlying liver dysfunction.

As used herein, “Budd-Chiari syndrome” (BCS) is defined as hepatic venous outflow tract obstruction, independent of the level or mechanism of obstruction, provided the obstruction is not due to cardiac disease, pericardial disease, or sinusoidal obstruction syndrome (veno-occlusive disease). Primary Budd-Chiari syndrome is present when there is obstruction due to a predominantly venous process (thrombosis or phlebitis), whereas secondary Budd-Chiari is present when there is compression or invasion of the hepatic veins and/or the inferior vena cava by a lesion that originates outside of the vein (e.g., a malignancy).

As used herein, “hepatitis” means an inflammation of the liver, irrespective of the cause. Hepatitis may be caused by a number of conditions, including drug toxicity, immune diseases, and viruses. It is characterized by jaundice, liver enlargement, and fever.

As used herein, a “kidney disease” is an acute or chronic damage to the kidney. It refers to a disease occurring in kidneys due to various reasons including extrinsic factors, intrinsic factors, genetic factors, etc. Non-limiting examples of renal diseases include: nephritis, nephrosis, renal fibrosis, thin glomerular basement membrane (TGBM), minimal change disease (MCD), membranous glomerulonephritis (MGN), focal segmental glomerulosclerosis (FSGS), DM nephropathy, IgA nephropathy (IgAN), tubulointerstitial nephritis (TIN), Henoch-Schonlein Purpura (HSP) nephritis, acute tubular injury, BK virus nephropathy, acute cellular rejection, chronic antibody mediated rejection, chronic active antibody mediated rejection, chronic calcineurin inhibitor toxicity, acute kidney injury, chronic kidney disease, ischemic renal disease, glomerulonephritis, lupus nephritis, polycystic kidney disease, pyelonephritis, nephrolith, renal tuberculosis, renal tumour, chronic renal failure, end stage renal failure, sepsis, renal injury caused by hepatic injury, etc.

As used herein, “acute kidney injury (AKI)” or “acute renal failure (ARF)” is an abrupt loss of kidney function that develops within 7 days. Non-limiting causes include: damage to the kidney tissue caused by decreased kidney blood flow (kidney ischemia) from any cause (e.g., low blood pressure), exposure to substances harmful to the kidney, an inflammatory process in the kidney, or an obstruction of the urinary tract that impedes the flow of urine. AKI is diagnosed on the basis of characteristic laboratory findings, such as elevated blood urea nitrogen and creatinine, or inability of the kidneys to produce sufficient amounts of urine. AKI may lead to a number of complications, including metabolic acidosis, high potassium levels, uremia, changes in body fluid balance, and effects on other organ systems, including death. People who have experienced AKI may have an increased risk of chronic kidney disease in the future.

As used herein, “chronic kidney disease (CKD)” means a type of kidney disease in which there is gradual loss of kidney function over a period of months or years. Early on there are typically no symptoms. Later, leg swelling, feeling tired, vomiting, loss of appetite, or confusion may develop. Complications may include heart disease, high blood pressure, bone disease, or anaemia. Non-limiting causes of chronic kidney disease include diabetes, high blood pressure, glomerulonephritis, and polycystic kidney disease. Risk factors include a family history of the condition. Diagnosis is generally by blood tests to measure the glomerular filtration rate and urine tests to measure albumin. Further tests such as an ultrasound or kidney biopsy may be performed to determine the underlying cause.

As used herein, “nephritis” means an inflammation of the kidneys and may involve the glomeruli, tubules, or interstitial tissue surrounding the glomeruli and tubules. Non-limiting causes include: infections, toxins and autoimmune disorders. Nephritis includes glomerulonephritis (inflammation of the glomeruli) and interstitial nephritis or tubulo-interstitial nephritis (inflammation of the spaces between renal tubules).

As used herein, “nephrosis” means any degenerative disease of the kidney tubules. Nephrosis can be caused by kidney disease, or it may be a complication of another disorder, particularly diabetes. The diagnosis is established via urine testing for the presence of protein, blood testing for lower-than-normal levels of protein, and observation of oedema.

As used herein, a “respiratory disease” means any pathological condition affecting the organs and tissues that make gas exchange possible in higher organisms, and includes conditions of the upper respiratory tract, trachea, bronchi, bronchioles, alveoli, pleura and pleural cavity, and the nerves and muscles of breathing.

As used herein, a “lung disease” is any respiratory disease affecting the lungs. A lung disease may be an acute or chronic damage to the lung. Non-limiting examples of lung diseases include: asthma, chronic obstructive pulmonary disease, chronic or acute bronchitis, cystic fibrosis emphysema, acute respiratory distress syndrome, bacterial pneumonia, tuberculosis pulmonary embolism and lung cancer.

As used herein, “pulmonary fibrosis” means a respiratory disease in which scars are formed in the lung tissues, leading to breathing problems. Scar formation, the accumulation of excess fibrous connective tissue (the process called fibrosis), leads to thickening of the walls and causes reduced oxygen supply in the blood. As a consequence, patients may suffer from shortness of breath. Symptoms of pulmonary fibrosis include: shortness of breath, chronic dry, hacking coughing, fatigue and weakness, chest discomfort including chest pain, loss of appetite and rapid weight loss. As disclosed herein, pulmonary fibrosis may be a secondary effect of other diseases and/or of specific treatments comprising a non-invasive administration for systemic and local delivery of therapeutic agents to the lungs, such as intranasal administration and oral inhalative administration. Non-liming examples of diseases and conditions that may cause pulmonary fibrosis as a secondary effect include: inhalation of environmental and occupational pollutants, such as metals in asbestosis, silicosis and exposure to certain gases; hypersensitivity pneumonitis, most often resulting from inhaling dust contaminated with bacterial, fungal, or animal products; cigarette smoking; some connective tissue diseases such as rheumatoid arthritis, Systemic lupus erythematosus (SLE) and scleroderma, sarcoidosis and granulomatosis with polyangiitis; infections; certain medications, e.g., amiodarone, bleomycin (pingyangmycin), busulfan, methotrexate, apomorphine and nitrofurantoin; radiation therapy to the chest.

The invention will now be described with reference to the following non-limiting Figures and Examples.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 . CNNM4 expression in liver determined by IHC in human samples and mouse models from DILI and different pathologies of chronic liver disease. *p<0.05 vs Healthy; **p<0.01 vs Healthy; ***p<0.001 vs Healthy.

FIG. 2A. CNNM4 expression, determined by qPCR of CNNM4 mRNA levels in human liver samples from healthy subjects compared with samples from steatotic and NASH patients.

FIG. 2B. CNNM4 expression, determined by qPCR of CNNM4 mRNA levels in an in vivo mouse model.

FIG. 2C. CNNM4 expression determined by qPCR of CNNM4 mRNA levels in an in vitro mouse cell model. *p<0.05 vs Healthy.

FIG. 3A. Lipid content in NASH-induced primary hepatocytes return into healthy levels when treated with siRNA CNNM4.

FIG. 3B. Inflammation induced by ROS in treated mice.

FIG. 3C. DILI-induced cell death is decreased by siRNA therapy. *p<0.05 vs Healthy **p<0.01 vs Healthy; ***p<0.001 vs Healthy; #p<0.05 vs NASH; ##p<0.01 vs NASH; ###p<0.001 vs DILI.

FIG. 4A. The lipid content in NASH-induced human cells returns to healthy levels when treated with CNNM4 siRNA.

FIG. 4B. The lipid content in NASH-induced human cells returns to healthy levels when treated with CNNM4 shRNA. *p<0.05 vs Healthy **p<0.01 vs Healthy; #p<0.05 vs NASH control; ##p<0.01 vs NASH control.

FIG. 5A. The lipid content in NASH-induced primary hepatocytes does not return to healthy levels when treated with siRNAs against CNNM1, CNNM2 or CNNM3.

FIG. 5B. Magnesium supplementation does not reduce lipid content in primary hepatocytes when CNNM4 is overexpressed.

FIG. 5C. CNNM4 siRNA treatment of primary hepatocytes reduces lipid accumulation caused by magnesium deficiency. ***p<0.001 vs Healthy; ##p<0.01 vs NASH model/Without Mg2+ + siRNA Ø.

FIG. 6 . Parameters analysed for observing NAFLD progression after CNNM4 siRNA therapy. A) Sudan Red as indicator of lipid content decrease, B) GPT in serum as indicator of liver damage, C) DHE as indicator of inflammation by ROS and D) αSMA as indicator of fibrosis. *p<0.05 vs siRNA Ø; **p<0.01 vs siRNA Ø.

FIG. 7 . Pharmacological inhibition of CNNM4 by 7-amino-2-phenyl-5H-thieno[3,2-c]pyridin-4-one. Treated hepatocytes have A) reduced lipid levels and B) an increase of intracellular magnesium levels. *p<0.05 vs Untreated; ***p<0.001 vs Untreated.

FIG. 8 . CNNM4 expression determined by IHC in animal samples of renal fibrosis. ***p<0.001 vs Healthy.

FIG. 9 . A) CNNM4 expression determined in TCGA (The Cancer Genome Atlas) primary tumour samples of Liver Hepatocellular Carcinoma (HHC) compared to normal tissue. B) CNNM4 expression determined in TCGA (The Cancer Genome Atlas) primary tumour samples of lung adenocarcinoma (LUAD) compared to normal tissue.

FIG. 10 . Possible synthesis route to DMT-Serinol(GaINAc)-CEP and CPG.

FIG. 11 . Reduction of human CNNM4 mRNA level in human HepG2 cells by transfection of CNNM4 siRNAs

FIG. 12 . Reduction of CNNM4 mRNA level in murine Hepa 1-6 cells by transfection of CNNM4 siRNAs.

FIG. 13 . Dose-dependent reduction of CNNM4 mRNA level in murine Hepa 1-6 cells by transfection of CNNM4 siRNAs

FIG. 14 . Inhibition of human CNNM4 gene expression in primary human hepatocytes by receptor mediated uptake of CNNM4 siRNA conjugates

FIG. 15 . Inhibition of mouse CNNM4 gene expression in primary mouse hepatocytes by receptor mediated uptake of CNNM4 siRNA conjugates.

FIG. 16 . Inhibition of human CNNM4 gene expression in primary human hepatocytes by receptor mediated uptake of CNNM4 siRNA conjugates.

FIG. 17 . Inhibition of CNNM4 gene expression in primary murine hepatocytes by receptor mediated uptake of CNNM4 siRNA conjugates.

FIG. 18 . Dose-dependent inhibition of CNNM4 target gene expression in the liver by CNNM4 siRNA conjugates.

FIG. 19 . Dose-dependent inhibition of CNNM4 target gene expression in the liver by CNNM4 siRNA conjugates.

FIG. 20 . Long-lasting inhibition of CNNM4 target gene expression in the liver by CNNM4 siRNA conjugates.

FIG. 21 . Inhibition of CNNM4 expression in rodent a NASH model treated with CNNM4 siRNA conjugates.

FIG. 22 . Treatment with CNNM4 siRNA conjugates reduces lipid accumulation in hepatocytes.

FIG. 23 . Treatment with CNNM4 siRNA conjugates reduces mitochondrial reactive oxygen species (ROS) production in hepatocytes.

FIG. 24 . Treatment of rodent NASH model with CNNM4 siRNA conjugates reduces development of NASH.

FIG. 25 . CNNM4 siRNA conjugates protect hepatocytes from apoptosis and cell death induced by acetaminophen (APAP).

FIG. 26 . Reduction of human CNNM4 mRNA level in human Huh-7 cells by transfection of CNNM4 siRNAs.

FIG. 27 . Dose-dependent reduction of CNNM4 mRNA level in human Huh-7 cells by transfection of CNNM4 siRNAs.

FIG. 28 . Inhibition of CNNM4 gene expression in primary human hepatocytes by receptor mediated uptake.

FIG. 29 . Inhibition of CNNM4 gene expression in primary cynomolgus hepatocytes by receptor mediated uptake.

FIG. 30 . Inhibition of CNNM4 gene expression in rodent model for NASH

EXAMPLES Example 1 Materials and Methods Human Samples

All the studies were performed in agreement with the Declaration of Helsinki and according to local national laws. The Human Ethics Committee of each hospital approved the study procedures and written informed consent was obtained from all patients before inclusion in the study.

Magnesium was quantified from human serum samples from a cohort of 8 healthy samples, 31 obese-diagnosed patients and 43 from a cohort of clinical trials. Patients were evaluated for non-alcoholic fatty liver disease (NAFLD) by different markers once discarded alcoholic disease and viral hepatitis infection.

Human CNNM4 expression in non-alcoholic fatty liver disease NAFLD was determined in a cohort of 42 patients: 10 healthy patients, 20 patients diagnosed with steatosis and 12 diagnosed with NAFLD. The CNNM4 levels in cirrhotic patients were determined in a cohort of 12 patients from which 5 were diagnosed as healthy and 7 as cirrhotic. 47 hepatocellular carcinoma (HCC) patients’ CNNM4 levels were also determined: 6 patients were healthy and 41 patients were diagnosed HCC. CNNM4 levels were determined in a cohort of 11 drug-induced liver injury (DILI) patients and compared to 3 healthy patients. Finally, renal fibrosis 14 human samples were analysed to determine CNNM4 expression. 7 samples were healthy and another 7 have been diagnoses with renal fibrosis.

Animal Experiments

All the animal experiments were conducted in accordance with the Spanish Guide for Care and use of Laboratory animals, and with the International Care and Use Committee Standards. All procedures were approved by the CIC bioGUNE’s Animal Care and Use Committee and the competent authority (Diputación de Bizkaia). Mice were housed in a temperature-controlled animal facility (AAALAC-accredited) within 12-hour light/dark cycles. G-They were fed a standard diet (Harlan Tekland) with water ad libitum.

NAFLD animal model: 0.1% methionine and choline-deficient diet (0.1% MCDD) for CNNM4 determination C57BL/6J wild-type mice were fed with a methionine (0.1%) and choline (0%) deficient diet for 4 weeks. At the end of the treatment animals were sacrificed and liver were split into several pieces for subsequent analysis including: RNA or protein extraction, formalin fixation for histology and immunohistochemistry or metabolic analysis. Blood for serum analysis was collected once a week during the treatment.

Pre-clinical Study: NAFLD Animal Model With siRNA Therapy

C57BL/6J wild-type mice were fed with a methionine (0.1%) and choline (0%) deficient diet for 4 weeks. 2 weeks after the beginning of the diet mice were divided in two groups and subjected to an in vivo silencing CNNM4 or unrelated siRNA control, receiving either 200 µll of a 0.75 µg/µl solution of or CNNM4-specific in vivo siRNA (Custom Ambion, USA) or control siRNA (Sigma-Aldrich, USA) using Invivofectamine ®3.0 Reagent (Invitrogen, USA) following the manufacturer’s instructions. Tail vein injection was performed twice a week until the fourth week. At the end of the treatment animals were sacrificed and liver were split into several pieces for subsequent analysis including: RNA or protein extraction, formalin fixation for histology and immunohistochemistry or metabolic analysis. Blood for serum analysis was collected once a week during the treatment.

Cirrhosis Animal Model: Bile Duct Ligation (BDL)

Adult C57BL/6J wilt-type mice were subjected to BDL as described previously (Fernandez-Alvarez et al., 2015. Lab Invest. 95(2):223-36). Briefly, mice were anesthetized with 1.5% isofluorane in O2 and the abdomen was opened. The bile duct was separated from the portal vein and the hepatic artery, performing a suture around the bile duct and securing with a surgical know. Finally, the abdomen was closed and mice sacrificed at 24 h, 48 h, 72 h, 3 days and 21 days. Liver were split into several pieces for subsequent analysis including: RNA or protein extraction, formalin fixation for histology and immunohistochemistry or metabolic analysis. Blood for serum analysis was collected once a week during the treatment.

Hepatocellular Carcinoma Animal Model (HCC): GNMT-/- Mice

Adult GNMT-/- mice were grown from 7 to 9 months, when they are described to develop spontaneously HCC (Wagner et al., 2009. Toxicol Appl Pharmacol. 1;237(2):246; author reply 247). At that time animals were sacrificed and liver were split into several pieces for subsequent analysis including: RNA or protein extraction, formalin fixation for histology and immunohistochemistry or metabolic analysis. Blood for serum analysis was collected once a week during the treatment.

Drug Induced Liver Injury (DILI): Acetaminophen (APAP) Overdose

Adult C57BL/6J wilt-type mice were treated with 500 mg/kg of acetaminophen (APAP) to induce acute liver injury. After 48 h of treatment, mice were sacrificed and liver were split into several pieces for subsequent analysis including: RNA or protein extraction, formalin fixation for histology and immunohistochemistry or metabolic analysis. Blood for serum analysis was collected once a week during the treatment.

Isolation of Primary Hepatocytes, Culture and Treatments

Primary hepatocytes from 3-month old wild-type (C57BL/6J) mice were isolated by perfusion with collagenase Type I (Worthington, USA). Briefly, animals were anesthetized with isoflurane (1.5% isoflurane in O2). Then, the abdomen was opened and a catheter was inserted into the inferior vena cava. Liver was perfused with buffer A (1x PBS, 5 mM EGTA, 37° C. and oxygenated) and the portal vein was cut. Next, liver was perfused with buffer B (1x PBS, 1 mM CaCl2 37° C. and oxygenated) to remove EGTA, and finally perfused with buffer C (1x PBS, 2 mM CaCl2, 0.65 BSA, collagenase type I, 37° C. and oxygenated). After buffer C perfusion, liver was separated from the resto of the body and placed into a petri dish with MEM (Gibco, USA). Gall bladder was carefully removed and, then, liver was mechanically disaggregated with forceps. The digested liver diluted in MEM was filtered through a sterile gauze and filtered liver cells were collected and washed three times (1x4′ at 400 RPM and 2x5′ at 500 RPM) in 10% FBS (Gibco)/1% PSG (Gibco) supplemented MEM, conserving all supernadant Kupffer and Hepatic Stellate cells isolation. After the final wash, hepatocytes contained in the pellet were resuspended in 10% FBS 1% PSG MEM for subsequently culturing.

Primary hepatocytes were seeded over previously collagen-coated culture dishes at a density of 7600 cells/mm2 in 10% FBS/1% PSG supplemented MEM and placed in an incubator at 37° C., 5%CO2-95% air. After 6 hours of attachment, culture medium and unattached hepatocytes were removed with fresh 0% FBS/1% PSG MEM for the aimed treatment.

THLE2 Cells

THLE-2 cells were purchased from ATCC (ATCC® CRL-2706TM). They were maintained on Bronchial Epithelial Growth Medium (BEGMTM, Lonza) supplemented with BEGM Bullet KitTM (Lonza) and 10%FBS. They were split with 0.05% trypsin-EDTA and collected in BEGM. After centrifugation at 123 g during 5 minutes, supernatant was discarded and pellet resuspended.

Plasmid Transfection

Plasmids were transfected into primary mouse hepatocytes for overexpression using jetPRIME® (Polyplus, USA) transfection reagent following manufacturer’s protocol. In a 24-well plate, 0.5 µg of plasmid were added to the jePRIME® buffer and vortexed 10s before adding 1 µljetPRIME® reagent. The mix was vortexed 10 s, spun down and incubated 10′ at RT). Transfections were performed in cell suspension medium and transfection mix was replaced for fresh medium 6 h after transfection unless indicated.

Gene Silencing by siRNA Delivery

Cells were transfected with specific siRNAs (Hs n7o for human CNNM4 and Mm n7o for mouse CNNM4) at a final concentration of 100 nM using DharmaFECT 1 reagent (Dharmacon) following manufacturer’s protocol. DharmaFECT 1 and siRNA were diluted separately in 0% FBS/1% PSG MEM for 5′ at RT. Dilutions were then mixed and incubated 20′ at RT. siRNA transfection mixes were then added to cell suspension medium and replaced for fresh medium after 6 h.

Gene Silencing by shRNA Delivery

Cells were transfected with a CNNM4 shRNA (SEQ ID NO: 454) by using lipofectamine® 3000 (Thermofisher) and following the protocol according manufacturer instructions. 7.5µl lipofectamine and the shRNA were diluted separately in 0.2ml culture medium and incubated during 5′ at room temperature. After incubation they were mixed again and incubated for 30′ at room temperature before delivering to the cells.

RNA Isolation

Total RNA from whole liver or cultured cells was isolated using TRIzol reagent (Invitrogen) according to manufacturer’s instruction. In case of cell mRNA extraction, 5 µg of Glycogen (Ambion, USA) were used in the RNA precipitation step to facilitate the visibility of the RNA pellet. RNA concentration was determined spectrophometrically using the Nanodrop ND-100 spectrophotometer (ThermoFisher Scientific, USA).

Retrotranscription

1-2 µg of isolated RNA were treated with DNAse I (Invitrogren) and used to synthesize cDNA by M-MLV reverse transcriptase in the presence of random primers and RNAseOUT (all from Invitrogen). Resulting cDNA was diluted ⅒ (1/20 if 2 µg were used) in RNAse free water (Sigma-Aldrich).

Real Time Quantitative PCR (RT-qPCR)

qPCRs were performed using either the ViiA 7 or the QS6 Real time PCR System with SYBR Select Master Mix (Applied Biosystems, USA). 1.5 µl of cDNA were used and including the specific primers for a total reaction volume of 6.5 µl in a 384-well plate (Applied Biosystems). All reactions were performed in triplicate. PCR conditions for the primers were optimized and 40 cycles with a melting temperature of 60° C. and 30 s per step were used. Both Homo Sapiens and Mus musculus primers were designed using the Primer 3 software via the NCBI-Nucleotide webpage (www.ncbi.nlm.nih.gov/nucleotide) and synthesized by Sigma Aldrich. After checking the specificity of the PCR products with the melting curve, data were normalized to the expression of a housekeeping gene (GAPDH, ARP).

Protein Extraction and Analysis

Extraction of total protein was performed as indicated. Cells were washed with cold PBS buffer and resuspended in 200 µl of RIPA lysis buffer (1.6 mM NaH2PO4, 8.4 mM Na2HPO4, 0.5% Azide, 0.1 M NaCl, 0.1% SDS, 0.1% Triton X-100, 5 mg/ml sodium deoxycholate). The lysis buffer was supplemented with protease and phosphatase inhibitor cocktails (Roche, Switzerland). They were centrifuged (13000 rpm, 20′ at 4° C.) and the supernatand (protein extract) was quantified for total protein content by the Bradford protein assay (Bio-Rad) and determined using a Spectramax M3 spectrophotometer (Molecular Devices, USA). In the case of frozen liver tissue, approximately 50 µg of tissue was homogenized by using a Precellys 24 tissue homogenizer (Precellys, France) in 500 µl of buffer. In all cases, the lysates were centrifuged (13000 rpm, 20 min, 4° C.) and the supernatant (protein extract) was quantified for total protein content by the Bradford protein assay or by BCA protein assay (Pierce, USA) depending on the type of lysis buffer used and determined using a Spectramax M3 spectrophotometer.

Western Blotting

Protein extracts were boiled at 95° C. for 5 min in SDS-PAGE sample buffer (250 mM Tris-HCl pH 6.8, 500 mM β-mercaptoethanol, 50% glycerol, 10% SDS and bromophenol blue). An appropriate amount of protein (between 5 and 50 µg), depending on protein abundance and antibody sensitivity, were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) in 3% to 15% acrylamide gels (depending on the molecular weight of the protein of interest), using a Mini-PROTEAN Electrophoresis System (Bio-Rad). Gels were transferred onto nitrocellulose membranes by electroblotting using a Mini Trans-Blot cell (Bio-Rad). Membranes were blocked with 5% nonfat milk in TBS pH 8 containing 0.1% Tween-20 (Sigma Aldrich) (TBST-0.1%), for 1 hour at RT, washed three times during 10′ with TBST-0.1% and incubated overnight at 4° C. with commercial primary antibodies. Primary antibodies and their optimal incubation conditions are detailed in Table 6. Membranes were then washed three times during 10′ with TBST-0.1% and incubated for 1 hour at RT in blocking solution containing secondary antibody conjugated to horseradish-peroxidase (HRP, Table 6). Immunoreactive proteins were detected by using Western Lightning Enhanced Chemiluminescence reagent (ECL, PerkinElmer, USA) and exposed to Super Rx-N X-ray films (Fuji, Japan) in a Curix 60 Developer (AGFA, Belgium).

Sudan Red for Lipid Staining

O.C.T-included frozen liver samples were cut into 10 µm sections. Sections were washed in 60% isopropanol and then stained with fresh Sudan III (0.5% in isopropanol; Sigma Aldrich) solution for 30 min. Samples were then washed again in 60% isopropanol and then counterstained with hematoxylin and eosin. The sections were then washed with distilled water and mounted in DPX mounting medium. Images were taken with an upright light microscope (Zeiss).

ROS Determination by DHE

O.C.T-embedded 8 µm sections were incubated with MnTBAP 150 µM 1h at RT. The samples were then incubated with dihydroethidium (DHE) 5 µM for 30 min at 37° C. and sections were mounted with Fluoromount-G (Southern Biotech, USA) containing 0.7 mg/l of DAPI to counterstain nuclei. Images were taken using an Axioimager D1 (Zeiss).

Immunohistochemistry for CNNM4 Determination

Paraffin-embedded sections (5 µm thick) were unmasked according to the primary antibody to be used and subjected to peroxide blocking (3% H2O2 in PBS, 10′, RT). For stainings with mouse-hosted antibodies in mouse tissues, samples were blocked with goat anti-mouse Fab fragment (Jackson Immunoresearch, USA) (1:10, 1 h, RT) and the blocked with 5% goat serum (30′, RT). Then, sections were incubated in a humid chamber with the CNNM4 primary antibody (Ab191207, Abcam) in DAKO antibody diluent (DAKO) at 1:100 followed by Envision anti rabbit (DAKO) HRP-conjugated secondary antibody incubation (30′, RT). Colorimetric detection was confirmed with Vector VIP chromogen (Vector) and sections were counterstained with hematoxylin. Samples were mounted using DPX mounting medium. Images were taken with an upright light microscope (Zeiss).

Immunofluorescence for αSMA Determination

For α-SMA staining, O.C.T-embedded 10 µm sections were incubated with a 1/200 dilution in 2%BSA in 0.01%PBS-azide of the primary antibody conjugated to Cy3 (C6198, Sigma Aldrich) and mounted with Fluoromount-G (Southern Biotech) containing 0.7 mg/l of DAPI to counterstain nuclei. Images were taken using an Axioimager D1 (Zeiss).

BODIPY for Lipid Quantification in Primary Hepatocytes

Primary hepatocytes cultured in high lipid content medium (OA) or methionine/choline deficient medium (MDMC) were fixed in 4% paraformaldehyde (10′, RT) in PBS and incubated with BODIPY 493/503 (Molecular Probes, Invitrogen) at 1 mg/ml (1h, RT). BODIPY immunocytofluorescence images were taken using an Axioimager D1 (Zeiss) microscope. Quantification of lipid bodies was performed using Frida Software and represented as mean area per total number of cells.

Data Analysis

The average sum of intensities or stained area percentage of each sample were calculated using the FRIDA software (http://bui3.win.ad.jhu.edu/frida/, John Hopkins University).

Liver Lipid Quantification

30 mg of frozen liver were homogenized with 10 volumes of ice-cold PBS in a potter homogenizer. Fatty acids were measured in the homogenates using the Wako Chemicals kit (Richmond, VA) and lipids were quantified as described (Folch et al., 1957. J Biol Chem. 226(1):497-509). Briefly, lipids were extracted from 1.5 mg of protein from liver homogenates. Phosphatidylcholine (PC), phosphatidylethanolamine (PE), fatty acids (FAs) and cholesterol (Ch) were separated by thin layer chromatography (TLC) and quantified as described (Ruiz and Ochoa, 1997). Triglycerides (TGs) were measured in the lipid extract with the A. Menarini Diagnostics (Italy) kit.

Intracellular Magnesium Levels

Primary hepatocytes grown in glass coverslips were loaded with 2 µM Mag-S-Tz or 1 µM Mag-S-Tz-AM diluted REF (Gruskos et al., 2016. J. Am. Chem. Soc. 138 (44), pp 14639-14649) in 0% FBS/1% PSG medium and incubated at 37° C. and 5% CO2 during 30′ or 1 h respectively. After removing the dye-containing medium, a 30′ incubation in 0% FBS/1% PSG was performed. Coverslips were then washed in a 20 mM Tris-HCl, 2.4 mM CaCl2, 10 mM glucose, pH 7.4 buffer and mounted on a perfusion chamber with thermostat on a Eclipse TE 300-based microspectrofluorometer (Nikon, USA) and visualized with a 40x oil-immersion fluorescence. Intracellular Mg2+ levels were determines using the method described by Grynkiewicz (Grynkiewiz et al., 1985. J Biol Chem. 260(6):3440-50). The 340/380 nm excited light ratio was determined with a Delta system (Photon Technologies International, Princeton) and converted into Mg²⁺ concentration from the standard equation:

$\left\lbrack {Mg^{2 +}} \right\rbrack i = \frac{\left( {R - R_{\min}} \right)}{\left( {R_{max} - R} \right)} \times K_{d} \times Q$

Where Kd is the Mg²⁺ dissociation constant of Mag-S-Tz (3.2 mM) and Mag-S-Tz-AM (8.9 mM) and Q is the ratio of the minimum/maximum fluorescence intensity at 380 nm.

Extracellular Magnesium Levels

Extracellular magnesium was quantified using the QuantiCromTM Magnesium Assay Kit (BioAssay Systems, USA). Briefly, 5 µl of serum or culture media were mixed with 200 µl of a 1:1 mix of Reagent A and Reagent B. After 2′ incubation at RT, OD was determined at 500 nm length using a Spectramax M3 spectrophotometer (Molecular Devices, USA). Then, 10 µl of EDTA were added and OD500 was determined again. Magnesium concentrations were finally calculated by comparing to the OD500 from a standard concentration (2 mg/ml).

Mitochondrial ROS Determination

Mitochondrial ROS was measured using MitoSOXTM Red reagent (Life Technologies), following manufacturer’s instructions. Briefly, primary hepatocytes and hepatoma cells were incubated with MitoSOX reagent (2.5 µM, 10′, 37° C. in a CO2 incubator) in normal culture medium. Then, cells were washed twice with PBS and the fluorescence was measured at an excitation of 510 nm and emission of 595 nm using a spectrophotometer. Final values were normalized to total protein concentration.

Cell Death Determination by TUNEL

Cell Death was analysed by using the In situ Cell Death detection Kit (Roche) following the manufacturer’s instructions as above indicated. Cells were subjected peroxide block (3% H2O2 in methanol) for 3 minutes before incubation with TUNEL diluent buffer containing FITC-conjugated primary antibody (dilution 1/50) for 1 hour at 37° C. Sections were mounted in Dako fluorescence mounting medium (Dako). Images were taken using an Axioimager D1 (Zeiss) microscope and cell viability was calculated by determining the % of TUNEL positive cells.

Results CNNM4 Overexpression in Liver Pathologies

Chronic liver disease includes a group of different pathologies. A method to detect CNNM4 expression by immunohistochemistry (IHC) in human liver biopsies and livers from mouse animal models has been developed. Herein CNNM4 expression has been characterized in DILI and all the stages from chronic liver disease, both in human biopsies and animal models, observing an overexpression of the protein in all the pathologies (FIG. 1 ). These results were confirmed by a method to detect CNNM4 expression by qPCR of CNNM4 mRNA levels in human liver samples from healthy subjects compared with samples from steatotic and NASH patients (FIG. 2A). CNNM4 expression could also be determined by qPCR of CNNM4 mRNA levels in an in vivo NASH mouse model (FIG. 2B), as well as in an in vitro NASH mouse cell model (FIG. 2C).

CNNM4 a New Target for Treating Liver Disease

The overexpression observed in CNNM4 determination by IHC suggests CNNM4 as a potential target for treating liver disease, both for DILI and chronic disease. An in vitro study was performed inducing NASH in primary hepatocytes and treating them with a siRNA CNNM4 therapy. In case of NASH model-primary hepatocytes the lipid content and inflammation by reactive oxygen species (ROS) were measured, observing that both get restored in NASH hepatocytes treated with the siRNA therapy (FIGS. 3A and 3B). A further in vitro study was performed on DILI induced by acetaminophen overdose. The expected cell death in the DILI model-hepatocytes was observed, as well as restoration upon siRNA treatment (FIG. 3C). A further in vitro study was performed in which NASH was induced in human cells and these cells were then treated with a siRNA CNNM4 therapy or with a shRNA CNNM4 therapy. Relative lipid accumulation restoration was observed both in NASH-induced human cells (THLE2 cells) treated with the siRNA therapy (FIG. 4A) and in NASH-induced human cells treated with the shRNA therapy (FIG. 4B).

Specificity and Need of Targeting CNNM4

Having observed the protective effect of siRNA CNNM4-therapies from NASH and DILI, the effect of targeted silencing the other proteins of CNNM family (CNNM1, CNNM2 and CNNM3) was determined. NASH was induced in primary hepatocytes and they were treated with siRNAs against CNNM1, CNNM2 and CNNM3. Differently than with siRNA CNNM4 therapy, silencing the other proteins of the CNNM family had no effect, indicating the specificity of a possible the treatment based only on CNNM4 and not on other CNNM family proteins (FIG. 5A). Furthermore, experiments were conducted which prove the need of a CNNM4-based treatment. In a first experiment, lipid accumulation was induced in primary hepatocytes by CNNM4 overexpression to mimic the situation observed in NASH patients and animal models. Magnesium supplementation had no effect on the level of lipid accumulation in those hepatocytes (FIG. 5B). In a second experiment, lipid accumulation was induced by magnesium deprivation in primary hepatocytes, which is leads to a similar physiological condition as CNNM4 overexpression. In this case, siRNA CNNM4 therapy reduced lipid accumulation (FIG. 5C). These last two results indicate that magnesium supplementation is not sufficient for treating NASH and that there is a need for a CNNM4-based therapy.

Preclinical Study of a siRNA CNNM4-based Therapy

A preclinical study in NAFLD, the first stage of chronic liver disease, was developed to test the efficacy of CNNM4 modulation not only in cells but also in animals. Mice were fed a 0.1% methionine and choline-deficient diet (0.1%MCDD) during two weeks in order for them to develop NAFLD. A subgroup was then treated with a siRNA CNNM4 therapy and another with a siCtrl RNA. Animals were sacrificed and different biomarkers were measured in order to analyse NAFLD progression. Sudan red staining was used to measure lipid accumulation (FIG. 6A). Transaminase levels in serum, which are indicative of liver damage, were also measured (FIG. 6B). DHE staining was used to quantify inflammation caused by ROS (FIG. 6C) and α-smooth-muscle actin (αSMA) levels were measured to indicate the progression of fibrosis (FIG. 6D). These results clearly indicate that siRNA CNNM4 therapy reduces NAFLD.

Pharmacological Inhibition of CNNM4

In addition to siRNA therapy, CNNM4 activity can be also modulated pharmacologically. An in vitro assay was performed inducing NASH in primary hepatocytes and treating them with the compound 7-amino-2-phenyl-5H-thieno[3,2-c]pyridin-4-one. It can be observed that this pharmacological inhibition of CNNM4 has the same effect as siRNA therapy in NASH-induced hepatocytes, it decreases its lipid content (FIG. 7A) and leads them to accumulate magnesium (FIG. 7B). It is therefore expected that a combination therapy of a CNNM4 siRNA and 7-amino-2-phenyl-5H-thieno[3,2-c]pyridin-4-one or one of its derivatives with similar function could lead to a potent treatment of NASH.

CNNM4 Over-Expression in Pathologies of Different Organs

As shown in FIG. 1 , CNNM4 is overexpressed in different liver pathologies, both in animal models and in human samples. However, fibrosis development occurs not only in liver but also in other organs such as the kidneys. There may be similarities in the development of fibrosis in both tissues and CNNM4 may also be deregulated in this organ. This is indeed the case. We observed a CNNM4 overexpression in a renal fibrosis mouse model, indicating that a therapy that reduces CNNM4 expression may also be effective in the treatment of kidney fibrosis (FIG. 8 ). In addition, an analysis of TCGA (The Cancer Genome Atlas) data show that CNNM4 is overexpressed in primary tumour samples of Liver Hepatocellular Carcinoma (HHC) and in primary tumour samples of lung adenocarcinoma (LUAD) compared to normal tissue (FIGS. 9A and 9B). Overexpression of CNNM4 has also been detected in cholangiocarcinoma (bile duct cancer). restore Mg²⁺ homeostasis

In summary, the presented results prove that CNNM4 is a suitable target for preventing NAFLD progression and indicates that inhibiting it could also ameliorate other liver pathologies (DILI, cirrhosis and HCC), renal fibrosis and lung cancer. CNNM4 is overexpressed in all these diseases. In addition, for DILI, the in vitro studies show a protective effect of siRNA therapy from APAP overdose. Inhibiting or silencing CNNM4, especially by siRNA therapy, is a suitable method for treating liver disease, renal fibrosis and lung cancer.

Example 2 - Synthesis of Building Blocks

The synthesis route for DMT-Serinol(GaINAc)-CEP and CPG as described below is outlined in FIG. 10 . Starting material DMT-Serinol(H) (1) was made according to literature published methods (Hoevelmann et al. Chem. Sci., 2016,7, 128-135) from commercially available L-Serine. GalNAc(Ac₃)-C₄H₈-COOH (2) was prepared according to literature published methods (Nair et al. J. Am. Chem. Soc., 2014, 136 (49), pp 16958-1696), starting from commercially available per-acetylated galactose amine. Phosphitylation reagent 2-Cyanoethyl-N,N-diisopropylchlorophosphor-amidite (4) is commercially available. Synthesis of (vp)-mU-phos was performed as described in Prakash, Nucleic Acids Res. 2015, 43(6), 2993-3011 and Haraszti, Nucleic Acids Res. 2017, 45(13), 7581-7592. Synthesis of the phosphoramidite derivatives of ST43 (ST43-phos) as well as ST23 (ST23-phos) can be performed as described in WO2017/174657.

DMT-Serinol(GalNAc) (3)

HBTU (9.16 g, 24.14 mmol) was added to a stirring solution of GalNAc(Ac₃)-C₄H₈-COOH (2) (11.4 g, 25.4 mmol) and DIPEA (8.85 ml, 50.8 mmol). After 2 minutes activation time a solution of DMT-Serinol(H) (1) (10 g, 25.4 mmol) in Acetonitrile (anhydrous) (200 ml) was added to the stirring mixture. After 1 h LCMS showed good conversion. The reaction mixture was concentrated in vacuo. The residue was dissolved up in EtOAc, washed subsequently with water (2x) and brine. The organic layer was dried over Na₂SO₄, filtered and concentrated under reduced pressure. The residue was further purified by column chromatography (3% MeOH in CH₂Cl₂+ 1% Et₃N, 700 g silica). Product containing fractions were pooled, concentrated and stripped with CH₂Cl₂ (2x) to yield to yield 10.6 g (51%) of DMT-Serinol(GalNAc) (3) as an off-white foam.

DMT-Serinol(GaINAc)-CEP (5)

2-Cyanoethyl-N,N-diisopropylchlorophosphoramidite (4) (5.71 ml, 25.6 mmol) was added slowly to a stirring mixture of DMT-Serinol(GalNAc) (3) (15.0 g, 17.0 mmol), DIPEA (14.9 ml, 85 mmol) and 4 Å molecular sieves in Dichloromethane (dry) (150 ml) at 0° C. under argon atmosphere. The reaction mixture was stirred at 0° C. for 1 h. TLC indicated complete conversion. The reaction mixture was filtered and concentrated in vacuo to give a thick oil. The residue was dissolved in Dichloromethane and was further purified by flash chromatography (0-50% acetone in toluene 1%Et3N, 220 g silica). Product containing fractions were pooled and concentrated in vacuo. The resulting oil was stripped with MeCN (2x) to yield 13.5 g (77%) of the colorless DMT-Serinol(GaINAc)-CEP (5) foam.

DMT-Serinol(GalNAc)-succinate (6)

DMAP (1.11 g, 9.11 mmol) was added to a stirring solution of DMT-Serinol(GalNAc) (3) (7.5 g, 9.11 mmol) and succinic anhydride (4.56 g, 45.6 mmol) in a mixture of Dichloromethane (50 ml) and Pyridine (50 ml) under argon atmosphere. After 16 h of stirring the reaction mixture was concentrated in vacuo and the residue was taken up in EtOAc and washed with 5% citric acid (aq). The aqueous layer was extracted with EtOAc. The combined organic layers were washed subsequently with sat NaHCO₃ (aq.) and brine, dried over Na₂SO₄, filtered and concentrated in vacuo. Further purification was achieved by flash chromatography (0-5% MeOH in CH₂Cl₂+1% Et₃N, 120 g silica). Product containing fractions were pooled and concentrated in vacuo. The residue was stripped with MeCN (3x) to yield 5.9 g (70%) DMT-Serinol(GalNAc)-succinate (6).

DMT-Serinol(GalNAc)-succinyl-lcaa-CPG (7)

The DMT-Serinol(GalNAc)-succinate (6) (1 eq.) and HBTU (1.1 eq.) were dissolved in CH₃CN (10 ml). Diisopropylethylamine (2 eq.) was added to the solution, and the mixture was swirled for 2 min followed by addition native amino-lcaa-CPG (500 A, 88 µmol/g, 1 eq.). The suspension was gently shaken at room temperature on a wrist-action shaker for 16 h, then filtered and washed with acetonitrile. The solid support was dried under reduced pressure for 2 h. The unreacted amines on the support were capped by stirring with Ac₂O/2,6-lutidine/NMl at room temperature (2x15 min). The washing of the support was repeated as above. The solid was dried under vacuum to yield DMT-Serinol(GalNAc)-succinyl-lcaa-CPG (7) (loading: 34 µmol/g, determined by detritylation assay).

Example 3 - Oligonucleotide Synthesis

Example compounds were synthesised according to methods described below and known to the person skilled in the art. Assembly of the oligonucleotide chain and linker building blocks was performed by solid phase synthesis applying phosphoramidite methodology.

Downstream cleavage, deprotection and purification followed standard procedures that are known in the art.

Oligonucleotide syntheses was performed on an AKTA oligopilot 10 using commercially available 2′O-Methyl RNA and 2′Fluoro-2′Deoxy RNA base loaded CPG solid support and phosphoramidites (all standard protection, ChemGenes, LinkTech) were used. Synthesis of DMT-(S)-Serinol(GalNAc)-succinyl Icaa CPG (7) and DMT-(S)-Serinol(GalNAC)-CEP (5) are described in example 2.

Ancillary reagents were purchased from EMP Biotech. Synthesis was performed using a 0.1 M solution of the phosphoramidite in dry acetonitrile (<20 ppm H₂O) and benzylthiotetrazole (BTT) was used as activator (0.3 M in acetonitrile). Coupling time was 10 min. A Cap/OX/Cap or Cap/Thio/Cap cycle was applied (Cap: Ac₂O/NMI/Lutidine/Acetonitrile, Oxidizer: 0.05 M I₂ in pyridine/H₂O). Phosphorothioates were introduced using commercially available thiolation reagent 50 mM EDITH in acetonitrile (Link technologies). DMT cleavage was achieved by treatment with 3% dichloroacetic acid in toluene. Upon completion of the programmed synthesis cycles a diethylamine (DEA) wash was performed. All oligonucleotides were synthesized in DMT-off mode.

Attachment of the Serinol(GalNAc) moiety was achieved by use of either base-loaded (S)-DMT-Serinol(GalNAc)-succinyl-lcaa-CPG (7) or a (S)-DMT-Serinol(GalNAc)-CEP (5). Tri-antennary GalNAc clusters (ST23/ST43) were introduced by successive coupling of the branching trebler amidite derivative (C6XLT-phos) followed by the GalNAc amidite (ST23-phos). Attachment of (vp)-mU moiety was achieved by use of (vp)-mU-phos in the last synthesis cycle. The (vp)-mU-phos does not provide a hydroxy group suitable for further synthesis elongation and therefore, does not possess an DMT-group. Hence coupling of (vp)-mU-phos results in synthesis termination.

For the removal of the methyl esters masking the vinylphosphonate, the CPG carrying the fully assembled oligonucleotide was dried under reduced pressure and transferred into a 20 ml PP syringe reactor for solid phase peptide synthesis equipped with a disc frit (Carl Roth GmbH). The CPG was then brought into contact with a solution of 250 µL TMSBr and 177 µL pyridine in CH₂Cl₂ (0.5 ml/µmol solid support bound oligonucleotide) at room temperature and the reactor was sealed with a Luer cap. The reaction vessels were slightly agitated over a period of 2x15 min, the excess reagent discarded, and the residual CPG washed 2x with 10 ml acetonitrile. Further downstream processing did not alter from any other example compound.

The single strands were cleaved off the CPG by 40% aq. methylamine treatment (90 min, RT). The resulting crude oligonucleotide was purified by ion exchange chromatography (Resource Q, 6 ml, GE Healthcare) on a AKTA Pure HPLC System using a sodium chloride gradient. Product containing fractions were pooled, desalted on a size exclusion column (Zetadex, EMP Biotech) and lyophilised until further use.

All final single-stranded products were analysed by AEX-HPLC to prove their purity. Identity of the respective single-stranded products was proved by LC-MS analysis.

Example 4 - Double-Strand Formation

Individual single strands were dissolved in a concentration of 60 OD/ml in H₂O. Both individual oligonucleotide solutions were added together in a reaction vessel. For easier reaction monitoring a titration was performed. The first strand was added in 25% excess over the second strand as determined by UV-absorption at 260 nm. The reaction mixture was heated to 80° C. for 5 min and then slowly cooled to RT. Double-strand formation was monitored by ion pairing reverse phase HPLC. From the UV-area of the residual single strand the needed amount of the second strand was calculated and added to the reaction mixture. The reaction was heated to 80° C. again and slowly cooled to RT. This procedure was repeated until less than 10% of residual single strand was detected.

Example 5

Reduction of human CNNM4 mRNA level in human HepG2 cells by transfection of CNNM4 siRNAs.

In vitro test shows reduction of CNNM4 mRNA levels in human HepG2 cells by transfection of CNNM4 siRNA molecules.

HepG2 cells were seeded in 96 well plates at a density of 40 000 cells per well with 10 nM siRNA and 0.3 µl RNAiFect added to the culture medium. The following day, cells were lysed for RNA extraction and CNNM4 and HPRT1 mRNA levels were determined by Taqman qRT-PCR. Values obtained for CNNM4 mRNA were normalized to values generated for the housekeeping gene HRPT1 and related to mean of untreated sample (ut) set at 1-fold target gene expression. Each bar represents mean +/- SD from three biological replicates. siRNA duplexes used in this study are listed in Table 2. Results are shown in FIGS. 11A and 11B.

Example 6

Reduction of CNNM4 mRNA level in murine Hepa 1-6 cells by transfection of CNNM4 siRNAs.

In vitro test shows reduction of CNNM4 mRNA levels in murine Hepa 1-6 cells by transfection of different CNNM4 siRNA molecules at a concentration of 10 nM.

Hepa 1-6 cells were seeded in 96 well plates at a density of 12 500 cells per well in the presence of 10 nM siRNA and 0.6 µl RNAiFect added to the culture medium. The following day, cells were lysed for RNA extraction and CNNM4 and ApoB mRNA levels were determined by Taqman qRT-PCR. Values obtained for CNNM4 mRNA were normalized to values generated for the housekeeping gene ApoB and related to mean of untreated sample (ut) set at 1-fold target gene expression. Each bar represents mean +/- SD from three biological replicates. siRNA duplexes used in this study are listed in Table 2. Results are shown in FIGS. 12A and 12B.

Example 7

Dose-dependent reduction of CNNM4 mRNA level in murine Hepa 1-6 cells by transfection of CNNM4 siRNAs.

In vitro test shows reduction of CNNM4 siRNAs mRNA levels in murine Hepa 1-6 cells by transfection of different CNNM4 siRNA molecules in a dose range of 4 nM to 0.0001 nM.

Hepa 1-6 cells were seeded in 96 well plates at a density of 12 500 cells per well in the presence of 4 nM, 0.8 nM, 0.16 nM, 0.032 nM, 0.006 nM, 0.001 nM, 0.0003 nM or 0.0001 nM siRNA and 0.6 µl RNAiFect added to the culture medium. The following day, cells were lysed for RNA extraction and CNNM4 and ApoB mRNA levels were determined by Taqman qRT-PCR. Values obtained for CNNM4 mRNA were normalized to values generated for the housekeeping gene ApoB and related to mean of untreated sample (ut) set at 1-fold target gene expression. Each bar represents mean +/- SD from three biological replicates. siRNA duplexes used in this study are listed in Table 2. Results are shown in FIGS. 13A to 13D.

Example 8

Inhibition of human CNNM4 gene expression in primary human hepatocytes by receptor mediated uptake of CNNM4 siRNA conjugates.

The example shows dose-dependent reduction of human CNNM4 mRNA levels by EU401 to EU414 in primary human hepatocytes.

Primary human hepatocytes (Life Technologies) were seeded in a 96 well plate at a density of 35 000 cells per well in plating medium and incubated with CNNM4 siRNA conjugates EU401 to EU414, at concentrations of 100 nM, 10 nM and 1 nM as shown in FIG. 14 , or they were incubated with non-targeting control conjugates (Ctr) at 100 nM (Ctr is EU400). The following day, cells were lysed for RNA extraction and CNNM4 and HPRT1 mRNA levels were determined by Taqman qRT-PCR. Values obtained for CNNM4 mRNA were normalized to values generated for the housekeeping gene HRPT1 and related to mean of untreated sample (ut) set at 1-fold target gene expression. Each bar represents mean +/- SD from three biological replicates. siRNA conjugates used in this study are listed in Table 2. Results are shown in FIG. 14 .

Example 9

Inhibition of mouse CNNM4 gene expression in primary mouse hepatocytes by receptor mediated uptake of CNNM4 siRNA conjugates.

The example shows dose-dependent reduction of mouse CNNM4 mRNA levels by EU401 to 408 and by EU410 to EU414 in primary mouse hepatocytes.

Primary mouse hepatocytes were seeded in a 96 well plate at a density of 25 000 cells per well in plating medium and incubated with CNNM4 siRNA conjugates EU401 to 408 and EU410 to EU414, at concentrations of 100 nM, 10 nM and 1 nM as shown in FIG. 15 , or they were incubated with non-targeting control conjugates (Ctr) at 100 nM (Ctr is EU400). The following day, cells were lysed for RNA extraction and CNNM4 and ApoB mRNA levels were determined by Taqman qRT-PCR. Values obtained for CNNM4 mRNA were normalized to values generated for the housekeeping gene ApoB and related to mean of untreated sample (ut) set at 1-fold target gene expression. Each bar represents mean +/- SD from three biological replicates. siRNA conjugates used in this study are listed in Table 2. Results are shown in FIG. 15 .

Example 10

Inhibition of human CNNM4 gene expression in primary human hepatocytes by receptor mediated uptake of CNNM4 siRNA conjugates.

The example shows dose-dependent reduction of human CNNM4 mRNA levels by EU415 to EU422 in primary human hepatocytes.

Primary human hepatocytes (Life Technologies) were seeded in a 96-well plate at a density of 35 000 cells per well in plating medium and incubated with CNNM4 siRNA conjugates EU415 to EU422 in concentrations of 100 nM, 10 nM and 1 nM as shown in FIG. 16 , or they were incubated with a non-targeting control conjugates (Ctr) at 100 nM (Ctr is EU423). The following day, cells were lysed for RNA extraction and CNNM4 and HPRT1 mRNA levels were determined by Taqman qRT-PCR. Values obtained for CNNM4 mRNA were normalized to values generated for the housekeeping gene HRPT1 and related to mean of untreated sample (ut) set at 1-fold target gene expression. Each bar represents mean +/- SD from three biological replicates. siRNA conjugates used in this study are listed in Table 2. Results are shown in FIG. 16 .

Example 11

Inhibition of CNNM4 gene expression in primary murine hepatocytes by receptor mediated uptake of CNNM4 siRNA conjugates.

The example shows dose-dependent reduction of mouse CNNM4 mRNA levels by EU415 to EU424 in primary mouse hepatocytes.

Primary mouse hepatocytes were seeded in a 96-well plate at a density of 25 000 cells per well in plating medium and incubated with CNNM4 siRNA conjugates EU415 to EU422, in concentrations of 100 nM, 10 nM and 1 nM as shown in FIG. 17 , or they were incubated with non-targeting control conjugates (Ctr) at 100 nM (Ctr is EU423). The following day, cells were lysed for RNA extraction and CNNM4 and ApoB mRNA levels were determined by Taqman qRT-PCR. Values obtained for CNNM4 mRNA were normalized to values generated for the housekeeping gene ApoB and related to mean of untreated sample (ut) set at 1-fold target gene expression. Each bar represents mean +/- SD from three biological replicates. siRNA conjugates used in this study are listed in Table 2. Results are shown in FIG. 17 .

Example 12

Dose-dependent inhibition of CNNM4 target gene expression in the liver by CNNM4 siRNA conjugates.

The example shows reduction of CNNM4 mRNA levels in the liver of wild-type mice two weeks after single dosing of EU403, EU404, EU408, EU412 and EU114 by subcutaneous injection.

Five- to seven-week old male C57BL/6 mice were treated with a single dose of 1 or 5 mg siRNA conjugate per kg body weight by subcutaneous injection. Control groups received a subcutaneous injection with the vehicle PBS. Two weeks after the treatment, liver samples were collected from all mice and snap frozen. RNA was extracted from liver samples and CNNM4 and Actin mRNA levels were determined by Taqman qRT-PCR. Values obtained for CNNM4 mRNA were normalized to values generated for the housekeeping gene Actin and related to mean of untreated sample (ut) set at 1-fold target gene expression. Each bar represents mean value from 6 animals +/-SD.

siRNA conjugates used in this study are listed in Table 2. The reduction of CNNM4 mRNA in mouse liver after treatment with siRNA conjugates is shown in FIG. 18 .

Example 13

Dose-dependent inhibition of CNNM4 target gene expression in the liver by CNNM4 siRNA conjugates.

The example shows reduction of CNNM4 mRNA levels in the liver of wild-type mice two weeks after single dosing of EU418, EU420 and EU422 by subcutaneous injection.

Five- to seven-week old male C57BL/6 mice were treated with a single dose of 0.3 or 1 mg siRNA conjugate per kg body weight by subcutaneous injection. Control groups received a subcutaneous injection with the vehicle PBS. Two weeks after the treatment, liver samples were collected from all mice and snap frozen. RNA was extracted from liver samples and CNNM4 and Actin mRNA levels were determined by Taqman qRT-PCR. Values obtained for CNNM4 mRNA were normalized to values generated for the housekeeping gene Actin and related to mean of untreated sample (ut) set at 1-fold target gene expression. Each bar represents mean value from 6 animals +/-SD.

siRNA conjugates used in this study are listed in Table 2. The reduction of CNNM4 mRNA in mouse liver after treatment with siRNA conjugates is shown in FIG. 19 .

Example 14

Long-lasting inhibition of CNNM4 target gene expression in the liver by CNNM4 siRNA conjugates.

The example shows reduction of CNNM4 mRNA levels in the liver of wild-type mice five weeks after a single dosing of EU418, EU420 and EU422 by subcutaneous injection.

Five- to seven-week old male C57BL/6 mice were treated with a single dose of 1 mg siRNA conjugate per kg body weight by subcutaneous injection. Control groups received a subcutaneous injection with the vehicle PBS. Five weeks after the treatment, liver samples were collected from all mice and snap frozen. RNA was extracted from liver samples and CNNM4 and Actin mRNA levels were determined by Taqman qRT-PCR. Values obtained for CNNM4 mRNA were normalized to values generated for the housekeeping gene Actin and related to mean of PBS treated cohort (PBS) set at 1-fold target gene expression. Each bar represents mean value from 6 animals +/-SD.

siRNA conjugates used in this study are listed in Table 2. The reduction of CNNM4 mRNA in mouse liver after treatment with siRNA conjugates is shown in FIG. 20 .

Example 15

Inhibition of CNNM4 expression in rodent a NASH model treated with CNNM4 siRNA conjugates.

The example shows reduction of CNNM4 mRNA levels in mice with NASH after treatment with a CNNM4 siRNA conjugate. The NASH phenotype was induced by feeding the mice with a diet devoid of choline and with 0.1% methionine for six weeks.

Three-month old male C57BL/6 mice were maintained on a diet deficient in choline with 0.1% methionine (0.1%MCDD) (A02082006i, Research Diets, Inc., New Jersey, USA) for six weeks. After three weeks of 0.1% MCDD, mice were treated with 1 mg or 5 mg siRNA per kg body weight of CNNM4 siRNA conjugate (EU414). Control groups received 1 mg/kg non-targeting siRNA conjugate (EU400). Mice were then maintained on 0.1% MCDD for another three weeks and liver samples were subsequently collected from all mice and snap frozen. RNA was extracted from liver samples and CNNM4 and Actin mRNA levels were determined by Taqman qRT-PCR. Values obtained for CNNM4 mRNA were normalized to values generated for the housekeeping gene Actin and related to mean of EU400 treated cohort set at 1-fold target gene expression. Each bar represents mean value from 6-8 animals +/-SD.

siRNA conjugates used in this study are listed in Table 2. The reduction of CNNM4 mRNA in the liver of murine NASH models three weeks after treatment with CNNM4 siRNA conjugate is shown in FIG. 21 .

Example 16

Treatment with CNNM4 siRNA conjugates reduces lipid accumulation in hepatocytes.

The example shows that lipid accumulation in hepatocytes is induced by oleic acid supplementation or by culturing the cells in methionine and choline deficient medium. Lipid accumulation is attenuated by treatment with CNNM4 siRNA conjugates.

Freshly isolated murine hepatocytes were seeded on coated cover slides in multi-well plates. Upon attachment, cells were incubated for 6 hours with 10 or 100 nM of EU403, EU404, EU408, EU412 or EU414. On the following day, the untreated cells (ut) were maintained in control medium (MEM/Gibco) for another 24 hours. The treated cells were cultured in the presence of 400 µM oleic acid (Sigma) or incubated with methionine- and choline- deficient DMEM/F12 medium (custom-made, Gibco) for 24 hours. Cells were then fixed in 4% paraformaldehyde solution. Lipid accumulation in hepatocytes was determined by staining of lipid bodies using boron-dipyromethene (BODIPY 493/503, Molecular Probes, Thermo Fisher Scientific). Images were acquired by fluorescence microscopy and BODIPY staining was quantified by ImageJ software.

Two independent experiments were carried out, each in quadruplicates. Values were normalized to mean values of untreated samples set at 1-fold. Each bar represents mean +/-SD. siRNA conjugates used in this study are listed in Table 2. The effect of CNNM4 siRNA treatment on lipid accumulation in methionine- and choline-deficient medium is shown in FIG. 22A. The effect of CNNM4 siRNA treatment on lipid accumulation induced by oleic acid is shown in FIG. 22B. Statistics: 2-way ANOVA with Dunnett’s post-hoc test against respective oleic acid and MCD control group on log-transformed values. * p≤ 0.05; **p ≤ 0.01;***p ≤ 001; ****p ≤ 0.0001. There are no significant inter-experimental differences.

Example 17

Treatment with CNNM4 siRNA conjugates reduces mitochondrial reactive oxygen species (ROS) production in hepatocytes.

The example shows that mitochondrial ROS induced by oleic acid supplementation or by culturing cells in methionine- and choline-deficient medium is reduced by treatment with CNNM4 siRNA molecules.

Freshly prepared murine hepatocytes were seeded in multi-well plates. Upon attachment, cells were incubated for 6 hours with 1 nM, 10 or 100 nM of EU404 or EU414. On the following day, the untreated cells (ut) were maintained in control medium (MEM/Gibco) for another 24 hours. The treated cells were cultured in the presence of 400 µM oleic acid (Sigma) or incubated with methionine- and choline-deficient DMEM/F12 medium (custom-made, Gibco BRL) for 24 hours. Mitochondrial ROS production in hepatocytes was assessed using MitoSOX Red mitochondrial superoxide indicator (Invitrogen, USA). The cells were loaded with 2 µM MitoSOX Red for 10 min at 37° C. in a CO₂ incubator. The cells were then washed three times with PBS. Fluorescence was measured at 510 nm (excitation) and 595 (emission) using a plate reader SpectraMax M2 (bioNova, USA).

Two independent experiments were each carried out in quadruplicates. Values were normalized to mean values of untreated samples set at 1-fold. Each bar represents mean +/-SD. siRNA conjugates used in this study are listed in Table 2. The effect of CNNM4 siRNA treatment on mitochondrial ROS production in methionine- and choline-deficient medium is shown in FIG. 23A. The effect of CNNM4 siRNA treatment on ROS production induced by oleic acid is shown in FIG. 23B. Statistics: 2-way ANOVA with Dunnett’s post-hoc test against respective oleic acid and MCD control group on log-transformed values. * p ≤ 0.05; **p ≤ 0.01;***p ≤ 001; ***p ≤ 0.0001; # indicates significant inter-experimental differences (p≤0.0001).

Example 18

Treatment of rodent NASH model with CNNM4 siRNA conjugates reduces development of NASH.

The example shows that treatment with a CNNM4 siRNA conjugate reduces lipid accumulation, reactive oxygen species (ROS) and fibrosis in a rodent NASH model. The NASH phenotype was induced by feeding the mice with a diet devoid of choline and with 0.1% methionine for six weeks.

Three-month old male C57BL/6 mice were maintained on a diet deficient in choline with 0.1% methionine (0.1%MCDD) (A02082006i, Research Diets, Inc., New Jersey, USA) for six weeks or fed a standard chow (SC). After three weeks of 0.1% MCDD, mice were treated with 1 mg or 5 mg CNNM4 siRNA conjugate (EU414) per body weight. Control groups fed with 0.1% MCD received 1 mg/kg non-targeting siRNA conjugate (EU400). Control mice fed with normal chow (SC) received the vehicle PBS at the same time point (three weeks). Mice were subsequently maintained on 0.1% MCDD or standard chow (SC) for another three weeks as indicated in FIG. 24 . Liver samples were collected from all mice at the end of the study and cryopreserved by embedding in optical coherence tomography cryocompound (OCT) or fixed with formalin and embedded in paraffin for preparation of sections. Lipid bodies and reactive oxygen species (ROS) were detected in cryosections by staining with Sudan red and Dihydroxyetidium (DHE), respectively. Liver fibrosis was assessed in paraffine sections by staining of the smooth muscle cell marker alpha smooth muscle actin (αSMA) by immunohistochemistry, as well as by detection of collagen fibers by Sirius red staining. Macrophages were detected by immunohistochemistry, by F4/80 staining of paraffin-embedded liver sections. Blood samples were collected from all animals at the six-week time point for serum preparation. Serum Mg²⁺ levels were then determined using the QuantiCrom™Magnesium Assay Kit (BioAssay Systems, USA).

siRNA conjugates used in this study are listed in Table 2. The reduction of liver steatosis by CNNM4 siRNA treatment in the 0.1% MCDD NASH model is shown in FIG. 24A. FIG. 24B shows reduction of reactive oxygen species by CNNM4 siRNA treatment. FIG. 24C shows the reduction of macrophage infiltration (F4/80 positive cells) in the liver by CNNM4 siRNA treatment. FIGS. 24D and 24E show the reduction in liver fibrosis by CNNM4 siRNA treatment in the same NASH model. These figures show quantifications of stained area percentages from each individual staining calculated using FIJI (https://imagej.net/Fiji.) in box and whisker plots with 5-95% percentile. n=5-7. (a.u. = arbitrary units). FIG. 24F shows reduction of serum Mg²⁺ levels in 0.1 %MCDD NASH model by treatment with EU414.

Example 19

CNNM4 siRNA conjugates protect hepatocytes from apoptosis and cell death induced by acetaminophen (APAP).

The example shows that inhibition of CNNM4 mRNA levels in primary hepatocytes with protects these cells from cell death induced by acetaminophen challenge.

Freshly prepared murine hepatocytes were seeded in multi-well plates. Upon attachment, cells were incubated for 6 hours with 1 nM and 10 nM of EU404 or EU414 or left untreated. Thereafter, cells were cultured in serum-free culture medium (MEM) for 13 to 14 hours. Subsequently, they remained untreated (Ut) or 10 mM acetaminophen was added to the medium (APAP). 6 hours later, cells were stained by Tunel assay or Trypan Blue staining to determine the proportion of cells that were necrotic (Tunel positive) or had undergone cell death (Trypan Blue positive).

siRNA conjugates used in this study are listed in Table 2. The reduction of CNNM4 expression by EU414 and EU404 is shown in FIG. 25A. The reduction of Tunel positive cells (necrosis marker) and Tripan Blue positive cells (cell death marker) by CNNM4 siRNA treatment after exposure to Acetaminophen is shown in FIGS. 25A and 25B, respectively.

Example 20

Reduction of CNNM4 mRNA level in human Huh-7 cells by transfection of CNNM4 siRNAs.

In vitro test shows reduction of CNNM4 mRNA levels in human Huh-7 cells by transfection of CNNM4 siRNA molecules.

Huh7 cells were seeded in 96 well plates at a density of 20 000 cells per well and transfected with 0.5 nM siRNA and RNAiMAX at a final concentration of 1 µl/ml culture medium. The following day, cells were lysed for RNA extraction and CNNM4 and HPRT1 mRNA levels were determined by Taqman qRT-PCR. Values obtained for CNNM4 mRNA were normalized to values generated for the house keeping gene HRPT1 and related to mean of untreated sample (ut) set at 1-fold target gene expression. Each bar represents mean +/- SD from three biological replicates. siRNA duplexes used in this study are listed in Table 2. Results are shown in FIG. 26 .

Example 21

Dose-dependent reduction of CNNM4 mRNA level in human Huh-7 cells by transfection of CNNM4 siRNAs.

In vitro test shows reduction of CNNM4 mRNA levels in human Huh-7 cells by transfection of different CNNM4 siRNA molecules in a dose range of 10 nM to 0.01 nM.

Huh-7 cells were seeded in the 96 well plate at a density of 20 000 cells per well and transfected with 10 nM, 1 nM, 0.1 nM, 0.01 nM siRNA and RNAiMAX at a final concentration of 1 µl/ml culture medium. The following day cells were lysed for RNA extraction and CNNM4 and HPRT1 mRNA levels were determined by Taqman qRT-PCR. Values obtained for CNNM4 mRNA were normalized to values generated for the house keeping gene HPRT1 and related to mean of untreated sample (ut) set at 1-fold target gene expression. Each bar represents mean +/- SD from three biological replicates. siRNA duplexes used in this study are listed in Table 2. Results are shown in FIGS. 27A and B.

Example 22

Inhibition of CNNM4 gene expression in primary human hepatocytes by receptor mediated uptake.

The example shows dose-dependent reduction of human CNNM4 mRNA levels by EU424 to EU433 in primary human hepatocytes by receptor mediated uptake.

Primary human hepatocytes (Life Technologies) were seeded in a 96 well plate at a density of 35 000 cells per well in plating medium and were subsequently incubated with CNNM4 siRNA conjugates EU424 to EU433 in concentrations of 100 nM, 10 nM, 1 nM, 0.1 nM or 0.01 nM or non-targeting control siRNA, EU423 (depicted as Ctr) as shown in FIG. 28 . Values obtained for CNNM4 mRNA were normalized to values generated for the house keeping gene HPRT1 and related to mean of untreated sample (ut) set at 1-fold target gene expression. Each bar represents mean +/- SD from three biological replicates. siRNA conjugates used in this study are listed in Table 2. Results with EU424 to EU433 are shown in FIG. 28 .

Example 23

Inhibition of CNNM4 gene expression in primary cynomolgus hepatocytes by receptor mediated uptake.

The example shows dose-dependent reduction of CNNM4 mRNA levels by EU429 to EU433 in primary cynomolgus hepatocytes by receptor mediated uptake.

Primary hepatocytes were seeded in a 96 well plate at a density of 45 000 cells per well in plating medium and were subsequently incubated with CNNM4 siRNA conjugates EU429 to EU433 in concentrations of 100 nM, 10 nM, 1 nM, 0.1 or 0.01 nM or 100 nM non-targeting control siRNA, EU423 (depicted as Ctr) as shown in FIG. 29 . Values obtained for CNNM4 mRNA were normalized to values generated for the house keeping gene PPIB and related to mean of untreated sample (ut) set at 1-fold target gene expression. Each bar represents mean +/- SD from three biological replicates. siRNA conjugates used in this study are listed in Table 2. Results with EU429 to 433 are shown in FIG. 29 .

Example 24

Inhibition of CNNM4 gene expression in rodent model for NASH.

The example shows dose dependent reduction of CNNM4 mRNA levels by EU422 in a murine model for NASH.

5-week old C57BL/6 male mice were maintained on a DIO-NASH diet for about 32 weeks and randomized based on pre-liver biopsy fibrosis and steatosis score. Only animals with fibrosis score of ≥ 1 and steatosis score of ≥ 2 were included in the study. Animals were then maintained on DIO-NASH diet for another 16 weeks. At the 4 week, 8 week, and 12 week time points after the pre liver biopsy mice were treated with 1 mg/kg or 5 mg/kg EU422, respectively or with the vehicle PBS by subcutaneous injection. 4 weeks after the last treatment, liver samples were collected from all mice and snap frozen. RNA was extracted from liver samples and CNNM4 and HPRT mRNA levels were determined by Taqman qRT-PCR. Values obtained for CNNM4 mRNA were normalized to values generated for the house keeping gene HPRT and related to mean of untreated sample (ut) set at 1-fold target gene expression. Each bar represents mean value from 15 animals +/-SD.

siRNA conjugates used in this study are listed in Table 2. The reduction of CNNM4 mRNA in mouse liver after treatment with siRNA conjugates is shown in FIG. 30 .

Summary Tables

TABLE 2 Summary duplex table Duplex Single Strands Duplex Single Strands Duplex Single Strands EU200 EU200A EU259 EU259A EU414 EU414A EU200B EU259B EU414B EU201 EU201A EU260 EU260A Mm n1 Mm n1 A EU201B EU260B Mm n1 B EU202 EU202A EU261 EU261A Mn n2 Mm n2 A EU202B EU261B Mm n2 B EU203 EU203A EU262 EU262A Mm n3 Mm n3 A EU203B EU262B Mm n3 B EU204 EU204A EU263 EU263A Mm n4 Mm n4 A EU204B EU263B Mm n4 B EU205 EU205A EU264 EU264A Mm n5 Mm n5 A EU205B EU264B Mm n5 B EU206 EU206A EU265 EU265A Mm n6 Mm n6 A EU206B EU265B Mn n6 B EU207 EU207A EU266 EU266A Hs n1 Hs n1 A EU207B EU266B Hs n1 B EU208 EU208A EU267 EU267A Hs n2 Hs n2 A EU208B EU267B Hs n2 B EU209 EU209A EU268 EU268A Hs n3 Hs n3 A EU209B EU268B Hs n3 B EU210 EU210A EU269 EU269A Hs n4 Hs n4 A EU210B EU269B Hs n4 B EU211 EU211A EU270 EU270A Hs n5 Hs n5 A EU211B EU270B Hs n5 B EU212 EU212A EU271 EU271A Hs n6 Hs n6 A EU212B EU271B Hs n6 B EU213 EU213A EU272 EU272A Hs n7 Hs n7 A EU213B EU272B Hs n7 B EU214 EU214A EU273 EU273A Mm n7 Mm n7 A EU214B EU273B Mm n7 B EU215 EU215A EU274 EU274A Hs n7o Hs n7 Ao EU215B EU274B Hs n7 Bo EU216 EU216A EU275 EU275A Mm n7o Mm n7 Ao EU216B EU275B Mm n7 Bo EU217 EU217A EU276 EU276A EU415 EU404A EU217B EU276B EU415B EU218 EU218A EU277 EU277A EU416 EU416A EU218B EU277B EU415B EU219 EU219A EU278 EU278A EU417 EU302A EU219B EU278B EU415B EU220 EU220A EU279 EU279A EU418 EU418A EU220B EU279B EU418B EU221 EU221A EU280 EU280A EU419 EU418A EU221B EU280B EU419B EU222 EU222A EU281 EU281A EU420 EU414A EU222B EU281B EU420B EU223 EU223A EU282 EU282A EU421 EU277A EU223B EU282B EU420B EU224 EU224A EU283 EU283A EU422 EU422A EU224B EU283B EU422B EU225 EU225A EU284 EU284A EU423 EU423A EU225B EU284B EU423B EU226 EU226A EU285 EU285A EU304 EU304A EU226B EU285B EU304B EU227 EU227A EU286 EU286A EU305 EU305A EU227B EU286B EU305B EU228 EU228A EU287 EU287A EU306 EU306A EU228B EU287B EU306B EU229 EU229A EU288 EU288A EU307 EU307A EU229B EU288B EU307B EU230 EU230A EU289 EU289A EU308 EU308A EU230B EU289B EU308B EU231 EU231A EU290 EU290A EU309 EU309A EU231B EU290B EU309B EU232 EU232A EU291 EU291A EU310 EU310A EU232B EU291B EU310B EU233 EU233A EU292 EU292A EU311 EU311A EU233B EU292B EU311B EU234 EU234A EU293 EU293A EU312 EU312A EU234B EU293B EU312B EU235 EU235A EU294 EU294A EU313 EU313A EU235B EU294B EU313B EU236 EU236A EU295 EU295A EU314 EU314A EU236B EU295B EU314B EU237 EU237A EU296 EU296A EU315 EU315A EU237B EU296B EU315B EU238 EU238A EU297 EU297A EU316 EU316A EU238B EU297B EU316B EU239 EU239A EU298 EU298A EU317 EU317A EU239B EU298B EU317B EU240 EU240A EU299 EU299A EU318 EU318A EU240B EU299B EU318B EU241 EU241A EU300 EU300A EU319 EU319A EU241B EU300B EU319B EU242 EU242A EU301 EU301A EU320 EU320A EU242B EU301B EU320B EU243 EU243A EU302 EU302A EU321 EU321A EU243B EU302B EU321B EU244 EU244A EU303 EU303A EU322 EU322A EU244B EU303B EU322B EU245 EU245A EU400 EU400A EU323 EU323A EU245B EU400B EU323B EU246 EU246A EU401 EU401A EU324 EU324A EU246B EU401B EU324B EU247 EU247A EU402 EU402A EU325 EU325A EU247B EU402B EU325B EU248 EU248A EU403 EU403A EU424 EU424A EU248B EU403B EU424B EU249 EU249A EU404 EU404A EU425 EU425A EU249B EU404B EU425B EU250 EU250A EU405 EU405A EU426 EU426A EU250B EU405B EU426B EU251 EU251A EU406 EU406A EU427 EU427A EU251B EU406B EU427B EU252 EU252A EU407 EU407A EU428 EU428A EU252B EU407B EU428B EU253 EU253A EU408 EU408A EU429 EU429A EU253B EU408B EU424B EU254 EU254A EU409 EU409A EU430 EU430A EU254B EU409B EU425B EU255 EU255A EU410 EU410A EU431 EU431A EU255B EU410B EU426B EU256 EU256A EU411 EU411A EU432 EU432A EU256B EU411B EU427B EU257 EU257A EU412 EU412A EU433 EU433A EU257B EU412B EU428B EU258 EU258A EU413 EU413A EU258B EU413B

TABLE 3 Summary abbreviations table Abbreviation Meaning mA, mU, mC, mG 2′-O-Methyl RNA nucleotides 2′-OMe 2′-O-Methyl modification fA, fU, fC, fG 2′ deoxy-2′-F RNA nucleotides 2′-F 2′-fluoro modification (ps) phosphorothioate (ps2) phosphorodithioate (vp) Vinyl-(E)-phosphonate (vp)-mU

(vp)-mU-phos

ivA, ivC, ivU, ivG inverted RNA (3′-3′) nucleotides ST23

ST23-phos

ST43 (or C6XLT)

ST43-phos (or C6XLT-phos)

Ser(GN) (when at the end of a chain, one of the O—— is OH)

[ST23(ps)]3 ST43(ps)

[ST23]3 ST43

[ST23(ps)]3 ST41 (ps)

[ST23]3 ST41

The abbreviations as shown in the above abbreviation table may be used herein. The list of abbreviations may not be exhaustive and further abbreviations and their meaning may be found throughout this document.

TABLE 4 Summary sequence table SEQ ID NO: Name (A=1^(st) strand; B=2^(nd) strand) Sequence 5′-3′ Unmodified sequence 5′-3′ counterpart 1 EU200A mU (ps) fc (ps) mGfAmAfGmUfAmUfUmCfCmGfCmGfUmLA (ps) fC (ps) mG UCGAAGUAUUCCGCGUACG 2 EU200B mC (ps) mG (ps) mUmAmCmGfCfGfGmAmAmUmAmCmUmUmC (ps) mG (ps) mA CGUACGCGGAAUACUUCGA 3 EU201A mC (ps) fC (ps) mUfAmGfGmAfUmCfAmCfGmCfUmCfUmG (ps) fC (ps) mU CCUAGGAUCACGCUCUGCU 4 EU201B mA (ps) mG (ps) mCmAmGmAfGfCfGmUmGmAmUmCmCmUmA (ps) mG (ps) mG AGCAGAGCGUGAUCCUAGG 5 EU202A mG (ps) fA (ps) mCfUmUfGmUfUmGfCmAfGmCfUmCfGmC (ps) fC (ps) mA GACUUGUUGCAGCUCGCCA 6 EU202B mU (ps) mG (ps) mGmCmGmAfGfCfUmGmCmAmAmCmAmAmG (ps) mU (ps) mC UGGCGAGCUGCAACAAGUC 7 EU203A mC (ps) fA (ps) mCfGmAfCmUfUmGfUmUfGmCfAmGfCmU (ps) fC (ps) mG CACGACUUGUUGCAGCUCG 8 EU203B mC (ps) mG (ps) mAmGmCmUfGfCfAmAmCmAmAmGmUmCmG (ps) mU (ps) mG CGAGCUGCAACAAGUCGUG 9 EU204A mG (ps) fA (ps) mCfAmCfGmAfAmGfAmUfGmAfUmGfCmC (ps) fA (ps) mU GACACGAAGAUGAUGCCAU 10 EU204B mA (ps) mU (ps) mGmGmCmAfUfCfAmUmCmUmUmCmGmUmG (ps) mU (ps) mC AUGGCAUCAUCUUCGUGUC 11 EU205A mC (ps) fG (ps) mGfUmGfAmAfGmGfAmGfAmUfCmAfGmG (ps) fU (ps) mU CGGUGAAGGAGAUCAGGUU 12 EU205B mA (ps) mA (ps) mCmCmUmGfAfUfCmUmCmCmUmUmCmAmC (ps) mC (ps) mG AACCUGAUCUCCUUCACCG 13 EU206A mC (ps) fU (ps) mCfAmCfGmUfUmGfAmCfCmAfGmCfUmG (ps) fC (ps) mU CUCACGUUGACCAGCUGCU 14 EU206B mA (ps) mG (ps) mCmAmGmCfUfGfGmUmCmAmAmCmGmUmG (ps) mA (ps) mG AGCAGCUGGUCAACGUGAG 15 EU207A mG (ps) fG (ps) mCfUmCfCmUfCmCfArnCfGmAfUmGfAmA (ps) fG (ps) mA GGCUCCUCCACCAUGAAGA 16 EU207B mU (ps) mC (ps) mUmUmCmAfUfGfGmUmGmGmAmGmGmAmG (ps) mC (ps) mC UCUUCAUGGUGGAGGAGCC 17 EU208A mG (ps) fA (ps) mAfAmAfUmAfUmGfCmCfCmGfAmCfAmG (ps) fC (ps)mA GAAAAUAUGCCCGACAGCA 18 EU208B mU (ps) mG (ps) mCmUmGmUfCfGfGmGmCmAmUmAmUmUmU (ps) mU (ps) mC UGCUGUCGGGCAUAUUUUC 19 EU209A mG (ps) fA (ps) mCfCmCfGmAfUmGfAmGfGmUfUmGfUmC (ps) fU (ps) mA GACCCGAUGAGGUUGUCUA 20 EU209B mU (ps) mA (ps) mGmAmCmAfAfCfCmUmCmAmUmCmGmGmG (ps) mU (ps) mC UAGACAACCUCAUCGGGUC 21 EU210A mG (ps) fG (ps) mAfCmCfCmGfAmUfGmAfGmGfUmUfGmU (ps) fC (ps) mU GGACCCGAUGAGGUUGUCU 22 EU210B mA (ps) mG (ps) mAmCmAmAfCfCfUmCmAmUmCmGmGmGmU (ps) mC (ps) mC AGACAACCUCAUCGGGUCC 23 EU211A mG (ps) fU (ps) mAfAmAfCmAfGmUfGmCfGmAfAmUfCmU (ps) fC (ps) mC GUAAACAGUGCGAAUCUCC 24 EU211B mG (ps) mG (ps) mAmGmAmUfUfCfGmCmAmCmUmGmUmUmU (ps) mA (ps) mC GGAGAUUCGCACUGUUUAC 25 EU212A mG (ps) fU (ps) mUfGmUfAmAfAmCfAmGfUmGfCmGfAmA (ps) fU (ps) mC GUUGUAAACAGUGCGAAUC 26 EU212B mG (ps) mA (ps) mUmUmCmGfCfAfCmUmGmUmUmUmAmCmA (ps) mA (ps) mC GAUUCGCACUGUUUACAAC 27 EU213A mC (ps) fA (ps) mUfUmAfUmAfGmGfGmCfUmCfUmGfUmC (ps) fA (ps) mC CAUUAUAGGGCUCUGUCAC 28 EU213B mG (ps) mU (ps) mGmAmCmAfGfAfGmCmCmCmUmAmUmAmA (ps) mU (ps) mG GUGACAGAGCCCUAUAAUG 29 EU214A mG (ps) fU (ps) mCfAmUfUmAfUmAfGmGfGmCfUmCfCmG (ps) fU (ps) mC GUCAUUAUAGGGCUCCGUC 30 EU214B mG (ps) mA (ps) mCmGmGmAfGfCfCmCmUmAmUmAmAmUmG (ps) mA (ps) mC GACGGAGCCCUAUAAUGAC 31 EU215A mG (ps) fA (ps) mUfCmAfUmAfUmUfGmAfGmCfUmCfCmU (ps) fC (ps) mU GAUCAUAUUGAGCUCCUCU 32 EU215B mA (ps) mG (ps) mAmGmGmAfGfCfUmCmAmAmUmAmUmGmA (ps) mU (ps) mC AGAGGAGCUCAAUAUGAUC 33 EU216A mG (ps) fC (ps)mUfGmGfGmUfCmAfUmGfAmUfAmUfCmC (ps) fU (ps) mC GCUGGGUCAUGAUAUCCUC 34 EU216B mG (ps) mA (ps) mGmGmAmUfAfUfCmAmUmGmAmCmCmCmA (ps) mG (ps) mC GAGGAUAUCAUGACCCAGC 35 EU217A mC (ps) fU (ps) mGfCmGfGmAfUmCfAmUfGmAfAmGfCmA (ps) fG (ps) mU CUGCGGAUCAUGAAGCAGU 36 EU217B mA (ps) mC (ps) mUmGmCmUfUfCfAmUmGmAmUmCmCmGmC (ps) mA (ps) mG ACUGCUUCAUGAUCCGCAG 37 EU218A mG (ps) fG (ps) mAfGmAfUmUfUmUfCmAfCmUfUmUfGmA (ps) fG (ps) mC GGAGAUUUUCACUUUGAGC 38 EU218B mG (ps) mC (ps) mUmCmAmAfAfGfUmGmAmAmAmAmUmCmU (ps) mC (ps) mC GCUCAAAGUGAAAAUCUCC 39 EU219A mC (ps) fU (ps) mUfAmUfUmUfCmGf GmGfUmGfUmAfCmA (ps) fG (ps) mG CUUAUUUCGGGUGUACAGG 40 EU219B mC (ps) mC (ps) mUmGmUmAfCfAfCmCmCmGmAmAmAmUmA (ps) mA (ps) mG CCUGUACACCCGAAAUAAG 41 EU220A mG (ps) fC (ps) mGfCmCfCmGfUmCfUmCfAmAfAmCfUmU (ps) fC (ps) mA GCGCCCGUCUCAAACUUCA 42 EU220B mU (ps) mG (ps) mAmAmGmUfUfUfGmAmGmAmCmGmGmGmC (ps) mG (ps) mC UGAAGUUUGAGACGGGCGC 43 EU221A mC (ps) fU (ps) mUfAmAfCmCfCmUfGmUfCmUfUmCfCmC (ps) fU (ps) mU CUUAACCCUGUCUUCCCUU 44 EU221B mA (ps) mA (ps) mGmGmGmAfAfGfAmCmAmGmGmGmUmUmA (ps) mA (ps) mG AAGGGAAGACAGGGUUAAG 45 EU222A mC (ps) fA (ps) mGfUmUfCmCfAmGfGmGfUmAfUmGfGmC (ps) fU (ps) mC CAGUUCCAGGGUAUGGCUC 46 EU222B mG (ps) mA (ps) mGmCmCmAfUfAfCmCmCmUmGmGmAmAmC (ps) mU (ps) mG GAGCCAUACCCUGGAACUG 47 EU223A mC (ps) fC (ps) mGfAmGfAmGfUmCfAmAfAmCfUmCfAmU (ps) fA (ps) mA CCGAGAGUCAAACUCAUAA 48 EU223B mU (ps) mU (ps) mAmUmGmAfGfUfUmUmGmAmCmUmCmUmC (ps) mG (ps) mG UUAUGAGUUUGACUCUCGG 49 EU224A mG (ps) fU (ps) mCfAmUfAmAfCmAfAmCfAmAfAmAfCmU (ps) fC (ps) mC GUCAUAACAACAAAACUCC 50 EU224B mG (ps) mG (ps) mAmGmUmUfUfUfGmUmUmGmUmUmAmUmG (ps) mA (ps) mC GGAGUUUUGUUGUUAUGAC 51 EU225A mC (ps) fC (ps) mAfUmAfUmAfUmCfGmUfUmUfAmGfCmA (ps) fA (ps) mA CCAUAUAUCGUUUAGCAAA 52 EU225B mU (ps) mU (ps) mUmGmCmUfAfAfAmCmGmAmUmAmUmAmU (ps) mG (ps) mG UUUGCUAAACGAUAUAUGG 53 EU226A mA (ps) fG (ps) mAfUmGfAmUfGmCfCmAfUmCfCmGfGmG (ps) fU (ps) mU AGAUGAUGCCAUCCGGGUU 54 EU226B mA (ps) mA (ps) mCmCmCmGfGfAfUmGmGmCmAmUmCmAmU (ps) mC (ps) mU AACCCGGAUGGCAUCAUCU 55 EU227A mA (ps) fC (ps) mGfAmAfGmAfUmGfAmUfGmCfCmAfUmC (ps) fC (ps) mG ACGAAGAUGAUGCCAUCCG 56 EU227B mC (ps) mG (ps) mGmAmUmGfGfCfAmUmCmAmUmCmUmUmC (ps) mG (ps) mU CGGAUGGCAUCAUCUUCGU 57 EU228A mU (ps) fC (ps) mGfGmAfCmAfCmGfAmAfGmAfUmGfAmU (ps) fG (ps) mC UCGGACACGAAGAUGAUGC 58 EU228B mG (ps) mC (ps) mAmUmCmAfUfCfUmUmCmGmUmGmUmCmC (ps) mG (ps) mA GCAUCAUCUUCGUGUCCGA 59 EU229A mA (ps) fA (ps) mGfUmUfCmAfCmCfGmUfGmCfUmGfCmC (ps) fC (ps) mU AAGUUCACCGUGCUGCCCU 60 EU229B mA (ps) mG (ps) mGmGmCmAfGfCfAmCmGmGmUmGmAmAmC (ps) mU (ps) mU AGGGCAGCACGGUGAACUU 61 EU230A mU (ps) fC (ps) mGfGmUfGmAfAmGfGmAfGmAfUmCfAmG (ps) fG (ps) mU UCGGUGAAGGAGAUCAGGU 62 EU230B mA (ps) mC (ps) mCmUmGmAfUfCfUmCmCmUmUmCmAmCmC (ps) mG (ps) mA ACCUGAUCUCCUUCACCGA 63 EU231A mA (ps) fC (ps) mCfUmCfGmGfUmGfAmAfGmGfAmGfAmU (ps) fC (ps) mA 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mUmCmAmAfCfAfCmCmUmCmCmCmUmCmA (ps) mC (ps) mA GGUCAACACCUCCCUCACA 75 EU237A mA (ps) fU (ps) mUfGmUfGmAfGmGfGmAfGmGfUmGfUmU (ps) fG (ps) mA AUUGUGAGGGAGGUGUUGA 76 EU237B mU (ps) mC (ps) mAmAmCmAfCfCfUmCmCmCmUmCmAmCmA (ps) mA (ps) mU UCAACACCUCCCUCACAAU 77 EU238A mU (ps) fC (ps) mUfAmGfAmAfGmGfAmUfUmGfUmGfAmG (ps) fG (ps) mG UCUAGAAGGAUUGUGAGGG 78 EU238B mC (ps) mC (ps) mCmUmCmAfCfAfAmUmCmCmUmUmCmUmA (ps) mG (ps) mA CCCUCACAAUCCUUCUAGA 79 EU239A mU (ps) fU (ps) mGfUmCfUmAfGmAfAmGfGmAfUmUfGmU (ps) fG (ps) mA UUGUCUAGAAGGAUUGUGA 80 EU239B mU (ps) mC (ps) mAmCmAmAfUfCfCmUmUmCmUmAmGmAmC (ps) mA (ps) mA UCACAAUCCUUCUAGACAA 81 EU240A mU (ps) fG (ps) mAfGmGfUmUfGmUfCmUfAmGfAmAfGmG (ps) fA (ps) mU UGAGGUUGUCUAGAAGGAU 82 EU240B mA (ps) mU (ps) mCmCmUmUfCfUfAmGmAmCmAmAmCmCmU (ps) mC (ps) mA AUCCUUCUAGACAACCUCA 83 EU241A mA (ps) fU (ps) mCfUmCfCmCfCmAfAmAfGmAfUmGfAmC (ps) fA (ps) mA AUCUCCCCAAAGAUGACAA 84 EU241B mU (ps) mU (ps) mGmUmCmAfUfCfUmUmUmGmGmGmGmAmG (ps) mA (ps) mU UUGUCAUCUUUGGGGAGAU 85 EU242A mA (ps) fG (ps) mAfAmCfAmAfUmAfGmUfGmUfUmGfGmC (ps) fA (ps) mC AGAACAAUAGUGUUGGCAC 86 EU242B mG(ps) mU (ps) mGmCmCmLAfAfCfAmCmUmAmUmUmGmUmU (ps) mC (ps) mU GUGCCAACACUAUUGUUCU 87 EU243A mA (ps) fG (ps) mCfAmUfAmAfAmGfAmAfUmUfUmGfGmU (ps) fG(ps) mA AGCAUAAAGAAUUUGGUGA 88 EU243B mU (ps) mC (ps) mAmCmCmAfAfAfUmUmCmUmUmUmAmUmG (ps) mC (ps) mU UCACCAAAUUCUUUAUGCU 89 EU244A mU (ps) fU (ps) mGfUmAfAmAfCmAfGmUfGmCfGmAfAmU (ps) fC (ps) mU UUGUAAACAGUGCGAAUCU 90 EU244B mA (ps) mG (ps) mAmUmUmCfGfCfAmCmUmGmUmUmUmAmC (ps) mA (ps) mA AGAUUCGCACUGUUUACAA 91 EU245A mA (ps) fC(ps) mAfGmUfUmUfUmGfGmUfCmCfUmUfAmG (ps) fU (ps) mU ACAGUUUUGGUCCUUAGUU 92 EU245B mA (ps) mA (ps) mCmUmAmAfGfGfAmCmCmAmAmAmAmCmU (ps) mG (ps) mU AACUAAGGACCAAAACUGU 93 EU246A mA (ps) fU (ps) mAfUmCfGmUfGmUfAmCfAmGfUmUfUmU (ps) fG (ps) mG AUAUCCUCUACAGUUUUGG 94 EU246B mC (ps) mC (ps) mAmAmAmAfCfUfGmUmAmGmAmGmGmAmU (ps) mA (ps) mU CCAAAACUGUAGAGGAUAU 95 EU247A mA (ps) fU (ps) mAfUmCfUmAfCmAfAmUfAmUfUmGfGmA (ps) fC (ps) mU AUAUCUACAAUAUUGGACU 96 EU247B mA (ps) mG (ps) mUmCmCmAfAfUfAmUmUmGmUmAmGmAmU (ps) mA (ps) mU AGUCCAAUAUUGUAGAUAU 97 EU248A mA (ps) fA (ps) mUfAmUfGmUfAmCfAmAfUmAfUmUfGmG (ps) fA (ps) mC AAUAUCUACAAUAUUGGAC 98 EU248B mG (ps) mU (ps) mCmCmAmAfUfAfUmUmGmUmAmGmAmUmA (ps) mU (ps) mU GUCCAAUAUUGUAGAUAUU 99 EU249A mU (ps) fU (ps) mGfAmCfGmUfAmGfAmGfAmAfUmAfUmC (ps) fU (ps) mA UUGACGUAGAGAAUAUCUA 100 EU249B mU (ps) mA (ps) mGmAmUmAfUfUfCmUmCmUmAmCmGmUmC (ps) mA (ps) mA UAGAUAUUCUCUACGUCAA 101 EU250A mA (ps) fA (ps) mGfUmCfUmUfUmGfAmCfGmUfAmGfAmG (ps) fA (ps) mA AAGUCUUUGACGUAGAGAA 102 EU250B mU (ps) mU (ps) mCmUmCmUfAfCfGmUmCmAmAmAmGmAmC (ps) mU (ps) mU UUCUCUACGUCAAAGACUU 103 EU251A mU (ps) fA (ps) mGfAmAfGmCfGmAfGmUfGmAfUmAfGmU (ps) fC (ps) mU UAGAAGCGAGUGAUAGUCU 104 EU251B mA (ps) mG (ps) mAmCmUmAfUfCfAmCmUmCmGmCmUmUmC (ps) mU (ps) mA AGACUAUCACUCGCUUCUA 105 EU252A mU (ps) fU (ps) mGfAmAfGmGfCmAfGmAfGmAfAmGfUmC (ps) fA (ps) mC UUGAAGGCAGAGAAGUCAC 106 EU252B mG (ps) mU (ps) mGmAmCmUfUfCfUmCmUmGmCmCmUmUmC (ps) mA (ps) mA GUGACUUCUCUGCCUUCAA 107 EU253A mU (ps) fU (ps) mUfCmAfCmUfUmUfGmAfmGmCfmUmCfAmU (ps) fU (ps) mG UUUCACUUUGAGCUCAUUG 108 EU253B mC (ps) mA (ps) mAmUmGmAfGfCfUmCmAmAmAmGmUmGmA (ps) mA (ps) mA CAAUGAGCUCAAAGUGAAA 109 EU254A mA (ps) fA (ps) mCfUmGfGmUfUmGfCmUfGmCfUmGfCmC (ps) fU (ps) mG AACUGGUUGCUGCUGCCUG 110 EU254B mC (ps) mA (ps) mGmGmCmAfGfCfAmGmCmAmAmCmCmAmG (ps) mU (ps) mU CAGGCAGCAGCAACCAGUU 111 EU255A mU (ps) fU (ps) mGfAmGfAmAfGmAfGmUfUmGfUmGfGmU (ps) fC (ps) mU UUGAGAAGAGUUGUGGUCU 112 EU255B mA (ps) mG (ps) mAmCmCmAfCfAfAmCmUmCmUmUmCmUmC (ps) mA (ps) mA AGACCACAACUCUUCUCAA 113 EU256A mU (ps) fU (ps) mCfAmUfGmUfGmGfGmCfCmCfAmCfUmG (ps) fG (ps) mG UUCAUGUGGGCCCACUGGG 114 EU256B mC (ps) mC (ps) mCmAmGmUfGfGfGmCmCmCmAmCmAmUmG (ps) mA (ps) mA CCCAGUGGGCCCACAUGAA 115 EU257A mU (ps) fC (ps) mUfUmCfAmUfGmUfGmGfGmCfCmCfAmC (ps) fU (ps) mG UCUUCAUGUGGGCCCACUG 116 EU257B mC (ps) mA (ps) mGmUmGmGfGfCfCmCmAmCmAmUmGmAmA (ps) mG (ps) mA CAGUGGGCCCACAUGAAGA 117 EU258A mU (ps) fG (ps) mGfAmCfUmAfAmCfAmGfGmUfUmCfCmC (ps) fU (ps) mC UGAACUAACAGGUUCCCUC 118 EU258B mG (ps) mA (ps) mGmGmGmAfAfCfCmUmGmUmUmAmGmUmC (ps) mC (ps) mA GAGGGAACCUGUUAGUCCA 119 EU259A mU (ps) fU (ps) mCfCmUfUmAfAmCfCmCfUmGfUmCfUmU (ps) fC (ps) mC UUCCUUAACCCUGUCUUCC 120 EU259B mG (ps) mG (ps) mAmAmGmAfCfAfGmGmGmUmUmAmAmGmG (ps) mA (ps) mA GGAAGACAGGGUUAAGGAA 121 EU260A mA (ps) fG (ps) mUfUmCfCmUfUmAfAmCfCmCfUmGfUmC (ps) fU (ps) mU AGUUCCUUAACCCUGUCUU 122 EU260B mA (ps) mA (ps) mGmAmCmAfGfGfGmUmUmAmAmGmGmAmA (ps) mC (ps) mU AAGACAGGGUUAAGGAACU 123 EU261A mA (ps) fU (ps) mAfAmAfGmUfUmCfCmUfUmAfAmCfCmC (ps) fU (ps) mG AUAAAGUUCCUUAACCCUG 124 EU261B mC (ps) mA (ps) mGmGmGmUfUfAfAmGmGmAmAmCmUmUmU (ps) mA (ps) mU CAGGGUUAAGGAACUUUAU 125 EU262A mA (ps) fA (ps) mAfUmAfAmAfGmUfUmCfCmUfUmAfAmC (ps) fC (ps) mC AAAUAAAGUUCCUUAACCC 126 EU262B mG (ps) mG (ps) mGmUmUmAfAfGfGmAmAmCmUmUmUmAmU (ps) mU (ps) mU GGGUUAAGGAACUUUAUUU 127 EU263A mU (ps) fA (ps) mCfAmUfAmAfAmGfUmUfCmCfUmUfAmA (ps) fC (ps) mC UACAUAAAGUUCCUUAACC 128 EU263B mG (ps) mG (ps) mUmUmAmAfGfGfAmAmCmUmUmUmAmUmG (ps) mU (ps) mA GGUUAAGGAACUUUAUGUA 129 EU264A mA (ps) fU (ps) mUfAmUfCmAfUmGfUmCfCmCfUmGfAmU (ps) fC (ps) mA AUUAUCAUGUCCCUGAUCA 130 EU264B mU (ps) mG (ps) mAmUmCmAfGfGfGmAmCmAmUmGmAmUmA (ps) mA (ps) mU UGAUCAGGGACAUGAUAAU 131 EU265A mA (ps) fU (ps) mAfAmAfGmAfAmCfCmUfGmCfUmUfCmC (ps) fG (ps) mU AUAAAGAACCUGCUUCCGU 132 EU265B mA (ps) mC (ps) mGmGmAmAfGfCfAmGmCmUmUmCmUmUmU (ps) mA (ps) mU ACGGAAGCAGGUUCUUUAU 133 EU266A mA (ps) fC (ps) mUfAmAfCmUfUmCfAmGfCmAfAmGfGmG (ps) fC (ps) mA ACUAACUUCAGCAAGGGCA 134 EU266B mU (ps) mG (ps) mCmCmCmUfUfGfCmUmGmAmAmGmUmUmA (ps) mG (ps) mU UGCCCUUGCUGAAGUUAGU 135 EU267A mU (ps) fG (ps) mGfUmGfCmAfAmGfAmCfAmAfUmCfCmC (ps) fU (ps) mG UGGUGCAAGACAAUCCCUG 136 EU267B mC (ps) mA (ps) mGmGmGmAfUfUfGmUmCmUmUmGmCmAmC (ps) mC (ps) mA CAGGGAUUGUCUUGCACCA 137 EU268A mA (ps) fU (ps) mAfAmAfUmAfGmGfAmGfUmCfAmUfCmU (ps) fG (ps) mC AUAAAUAGGAGUCAUCUGC 138 EU268B mG (ps) mC (ps) mAmGmAmUfGfAfCmUmCmCmUmAmUmUmU (ps) mA (ps) mU GCAGAUGACUCCUAUUUAU 139 EU269A mU (ps) fU (ps) mGfGmAfAmAfUmAfAmUfGmAfUmGfAmC (ps) fC (ps) mC UUGGAAAUAAUGAUGACCC 140 EU269B mG (ps) mG (ps) mGmUmCmAfUfCfAmUmUmAmUmUmUmCmC (ps) mA (ps) mA GGGUCAUCAUUAUUUCCAA 141 EU270A mA (ps) fA (ps) mAfCmUfCmAfUmAfAmCfAmAfUmGfCmC (ps) fA (ps) mA AAACUCAUAACAAUGCCAA 142 EU270B mU (ps) mU (ps) mGmGmCmAfUfUfGmUmUmAmUmGmAmGmU (ps) mU (ps) mU UUGGCAUUGUUAUGAGUUU 143 EU271A mA (ps) fU (ps) mUfAmUfCmAfUmUfGmUfUmUfUmGfUmG (ps) fA (ps) mC AUUAUCAUUGUUUUGUGAC 144 EU271B mG (ps) mU (ps) mCmAmCmAfAfAfAmCmAmAmUmGmAmUmA (ps) mA (ps) mU GUCACAAAACAAUGAUAAU 145 EU272A mU (ps) fU (ps) mGfUmUfUmAfGmCfAmAfAmUfAmUfUmA (ps) fU (ps) mC UUGUUUAGCAAAUAUUAUC 146 EU272B mG (ps) mA (ps) mUmAmAmUfAfUfUmUmGmCmUmAmAmAmC (ps) mA (ps) mA GAUAAUAUUUGCUAAACAA 147 EU273A mU (ps) fC (ps) mCfCmCfGmAfGmAfGmUfCmAfAmAfCmU (ps) fC (ps) mA UCCCCGAGAGUCAAACUCA 148 EU273B mU (ps) mG (ps) mAmGmUmUfUfGfAmCmUmCmUmCmGmGmG (ps) mG (ps) mA UGAGUUUGACUCUCGGGGA 149 EU274A mA (ps) fA (ps) mCfUmCfCmCfCmGfAmGfAmGfUmCfAmA (ps) fA (ps) mC AACUCCCCGAGAGUCAAAC 150 EU274B mG (ps) mU (ps) mUmUmGmAfCfUfCmUmCmGmGmGmGmAmG (ps) mU (ps) mU GUUUGACUCUCGGGGAGUU 151 EU275A mA (ps) fA (ps) mCfAmAfCmAfAmAfAmCfUmCfCmCfCmG (ps) fA (ps) mG AACAACAAAACUCCCCGAG 152 EU275B mC (ps) mU (ps) mCmGmGmGfGfAfGmUmUmUmUmGmUmUmG (ps) mU (ps) mU CUCGGGGAGUUUUGUUGUU 153 EU276A mA (ps) fU (ps) mAfAmCfAmAfCmAfAmAfAmCfUmCfCmC (ps) fC (ps) mG AUAACAACAAAACUCCCCG 154 EU276B mC (ps) mG (ps) mGmGmGmAfGfUfUmUmUmGmUmUmGmUmU (ps) mA (ps) mU CGGGGAGUUUUGUUGUUAU 155 EU277A mU (ps) fG (ps) mAfCmAfAmAfAmGfAmCfAmCfAmAfGmA (ps) fG (ps) mU UGACAAAAGACACAAGAGU 156 EU277B mA (ps) mC (ps) mUmCmUmUfGfUfGmUmCmUmUmUmUmGmU (ps) mC (ps) mA ACUCUUGUGUCUUUUGUCA 157 EU278A mU (ps) fU (ps) mGfUmUfUmUfGmUfGmAfCmAfAmAfAmG (ps) fA (ps) mC UUGUUUUGUGACAAAAGAC 158 EU278B mG (ps) mU (ps) mCmUmUmUfUfGfUmCmAmCmAmAmAmAmC (ps) mA (ps) mA GUCUUUUGUCACAAAACAA 159 EU279A mU (ps) fC (ps) mUfAmGfGmAfUmCfAmCfGmCfUmCfUmG (ps) fC (ps) mU UCUAGGAUCACGCUCUGCU 160 EU279B mA (ps) mG (ps) mCmAmGmAfGfCfGmUmGmAmUmCmCmUmA (ps) mG (ps) mA AGCAGAGCGUGAUCCUAGA 161 EU280A mU (ps) fA (ps) mCfUmUfGmUfUmGfCmAfGmCfUmCfGmC (ps) fC (ps) mA UACUUGUUGCAGCUCGCCA 162 EU280B mU (ps) mG (ps) mGmCmGmAfGfCfUmGmCmAmAmCmAmAmG (ps) mU (ps) mA UGGCGAGCUGCAACAAGUA 163 EU281A mU (ps) fA (ps) mCfGmAfCmUfUmGfUmUf GmCfAmGfCmU (ps) fC (ps) mG UACGACUUGUUGCAGCUCG 164 EU281B mC (ps) mG (ps) mAmGmCmUfGfCfAmAmCmAmAmGmUmCmG (ps) mU (ps) mA CGAGCUGCAACAAGUCGUA 165 EU282A mU (ps) fA (ps) mCfAmCfGmAfAmGfAmUfGmAfUmGfCmC (ps) fA (ps) mU UACACGAAGAUGAUGCCAU 166 EU282B mA (ps) mU (ps) mGmGmCmAfUfCfAmUmCmUmUmCmGmUmG (ps) mU (ps) mA AUGGCAUCAUCUUCGUGUA 167 EU283A mU (ps) fG (ps) mGfUmGfAmAfGmGfAmGfAmUfCmAfGmG (ps) fU (ps) mU UGGUGAAGGAGAUCAGGUU 168 EU283B mA (ps) mA (ps) mCmCmUmGfAfUfCmUmCmCmUmUmCmAmC (ps) mC (ps) mA AACCUGAUCUCCUUCACCA 169 EU284A mU (ps) fU (ps) mCfAmCfGmUfUmGfAmCfCmAfGmCfUmG (ps) fC (ps) mU UUCACGUUGACCAGCUGCU 170 EU284B mA (ps) mG (ps) mCmAmGmCfUfGfGmUmCmAmAmCmGmUmG (ps) mA (ps) mA AGCAGCUGGUCAACGUGAA 171 EU285A mU (ps) fG (ps) mCfUmCfCmUfCmCfAmCfCmAfUmGfAmA (ps) fG (ps) mA UGCUCCUCCACCAUGAAGA 172 EU285B mU (ps) mC (ps) mUmUmCmAfUfGfGmUmGmGmAmGmGmAmG (ps) mC (ps) mA UCUUCAUGGUGGAGGAGCA 173 EU286A mU (ps) fA (ps) mAfAmAfUmAfUmGfCmCfCmGfAmCfAmG (ps) fC (ps) mA UAAAAUAUGCCCGACAGCA 174 EU286B mU (ps) mG (ps) mCmUmGmUfCfGfGmGmCmAmUmAmUmUmU (ps) mU (ps) mA UGCUGUCGGGCAUAUUUUA 175 EU287A mU (ps) fA (ps) mCfCmCfGmAfUmGfAmGfGmUfUmGfUmC (ps) fU (ps) mA UACCCGAUGAGGUUGUCUA 176 EU287B mU (ps) mA (ps) mGmAmCmAfAfCfCmUmCmAmUmCmGmGmG (ps) mU (ps) mA UAGACAACCUCAUCGGGUA 177 EU288A mU (ps) fG (ps) mAfCmCfCmGfAmUfGmAfGmGfUmUfGmU (ps) fC (ps) mU UGACCCGAUGAGGUUGUCU 178 EU288B mA (ps) mG (ps) mAmCmAmAfCfCfUmCmAmUmCmGmGmGmU (ps) mC (ps) mA AGACAACCUCAUCGGGUCA 179 EU289A mU (ps) fU (ps) mAfAmAfCmAfGmUfGmCfGmAfAmUfCmU (ps) fC (ps) mC UUAAACAGUGCGAAUCUCC 180 EU289B mG (ps) mG (ps) mAmGmAmUfUfCfGmCmAmCmUmGmUmUmU (ps) mA (ps) mA GGAGAUUCGCACUGUUUAA 181 EU290A mU (ps) fU (ps) mUfGmUfAmAfAmCfAmGfUmGfCmGfAmA (ps) fU (ps) mC UUUGUAAACAGUGCGAAUC 182 EU290B mG (ps) mA (ps) mUmUmCmGfCfAfCmUmGmUmUmUmAmCmA (ps) mA (ps) mA GAUUCGCACUGUUUACAAA 183 EU291A mU (ps) fA (ps) mUfUmAfUmAfGmGfGmCfUmCfUmGfUmC (ps) fA (ps) mC UAUUAUAGGGCUCUGUCAC 184 EU291B mG (ps) mU (ps) mGmAmCmAfGfAfGmCmCmCmUmAmUmAmA (ps) mU (ps) mA GUGACAGAGCCCUAUAAUA 185 EU292A mU (ps) fU (ps) mCfAmUfUmAfUmAfGmGfGmCfUmCfCmG (ps) fU (ps) mC UUCAUUAUAGGGCUCCGUC 186 EU292B mG (ps) mA (ps) mCmGmGmAfGfCfCmCmUmAmUmAmAmUmG (ps) mA (ps) mA GACGGAGCCCUAUAAUGAA 187 EU293A mU (ps) fA (ps) mUfCmAfUmAfUmUfGmAfGmCfUmCfCmU (ps) fC (ps) mU UAUCAUAUUGAGCUCCUCU 188 EU293B mA (ps) mG (ps) mAmGmGmAfGfCfUmCmAmAmUmAmUmGmA (ps) mU (ps) mA AGAGGAGCUCAAUAUGAUA 189 EU294A mU (ps) fC (ps) mUfGmGfGmUfCmAfUmGfAmUfAmUfCmC (ps) fU (ps) mC UCUGGGUCAUGAUAUCCUC 190 EU294B mG (ps) mA (ps) mGmGmAmUfAfUfCmAmUmGmAmCmCmCmA (ps) mG (ps) mA GAGGAUAUCAUGACCCAGA 191 EU295A mU (ps) fU (ps) mGfCmGfGmAfUmCfAmUfGmAfAmGfCmA (ps) fG (ps) mU UUGCGGAUCAUGAAGCAGU 192 EU295B mA (ps) mC (ps) mUmGmCmUfUfCfAmUmGmAmUmCmCmGmC (ps) mA (ps) mA ACUGCUUCAUGAUCCGCAA 193 EU296A mU (ps) fG (ps) mAfGmAfUmUfUmUfCmAfCmUfUmUfGmA (ps) fG (ps) mC UGAGAUUUUCACUUUGAGC 194 EU296B mG (ps) mC (ps) mUmCmAmAfAfGfUmGmAmAmAmAmUmCmU (ps) mC (ps) mA GCUCAAAGUGAAAAUCUCA 195 EU297A mU (ps) fU (ps) mUfAmUfUmUfCmGfGmGfUmGfUmAfCmA (ps) fG (ps) mG UUUAUUUCGGGUGUACAGG 196 EU297B mC (ps) mC (ps) mUmGmUmAfCfAfCmCmCmGmAmAmAmUmA (ps) mA (ps) mA CCUGUACACCCGAAAUAAA 197 EU298A mU (ps) fC (ps) mGfCmCfCmGfUmCfUmCfAmAfAmCfUmU (ps) fC (ps) mA UCGCCCGUCUCAAACUUCA 198 EU298B mU (ps) mG (ps) mAmAmGmUfUfUfGmAmGmAmCmGmGmGmC (ps) mG (ps) mA UGAAGUUUGAGACGGGCGA 199 EU299A mU (ps) fU (ps) mUfAmAfCmCfCmUfGmUfCmUfUmCfCmC (ps) fU (ps) mU UUUAACCCUGUCUUCCCUU 200 EU299B mA (ps) mA (ps) mGmGmGmAfAfGfAmCmAmGmGmGmUmUmA (ps) mA (ps) mA AAGGGAAGACAGGGUUAAA 201 EU300A mU (ps) fA (ps) mGfUmUfCmCfAmGfGmGfUmAfUmGfGmC (ps) fU (ps) mC UAGUUCCAGGGUAUGGCUC 202 EU300B mG (ps) mA (ps) mGmCmCmAfUfAfCmCmCmUmGmGmAmAmC (ps) mU (ps) mA GAGCCAUACCCUGGAACUA 203 EU301A mU (ps) fC (ps) mGfAmGfAmGfUmCfAmAfAmCfUmCfAmU (ps) fA (ps) mA UCGAGAGUCAAACUCAUAA 204 EU301B mU (ps) mU (ps) mAmUmGmAfGfUfUmUmGmAmCmUmCmUmC (ps) mG (ps) mA UUAUGAGUUUGACUCUCGA 205 EU302A mU (ps) fU (ps) mCfAmUfAmAfCmAfAmCfAmAfAmAfCmU (ps) fC (ps) mC UUCAUAACAACAAAACUCC 206 EU302B mG (ps) mG (ps) mAmGmUmUfUfUfGmUmUmGmUmUmAmUmG (ps) mA (ps) mA GGAGUUUUGUUGUUAUGAA 207 EU303A mU (ps) fC (ps) mAfUmAfUmAfUmCfGmUfUmUfAmGfCmA (ps) fA (ps) mA UCAUAUAUCGUUUAGCAAA 208 EU303B mU (ps) mU (ps) mUmGmCmUfAfAfAmCmGmAmUmAmUmAmU (ps) mG (ps mA UUUGCUAAACGAUAUAUGA 209 EU400A mU (ps) fC (ps) mGfAmAfGmUfAmUfUmCfCmGfCmGfUmA (ps) fC (ps) mG UCGAAGUAUUCCGCGUACG 210 EU400B [ST23 (ps) ] 3ST43 (ps) mCmGmUmAmCmGfCfGfGmAmAmUmAmCmUmUmC (ps) mG (ps) mA CGUACGCGGAAUACUUCGA 211 EU401A (vp) mUfAmCfGmAfCmUfUmGfUmUfGmCfAmGfCmU (ps) fC (ps) mG UACGACUUGUUGCAGCUCG 212 EU401B [ST23 (ps) ] 3ST43 (ps) mCmGmAmGmCmUfGfCfAmAmCmAmAmGmUmCmG (ps) mU (ps) mG CGAGCUGCAACAAGUCGUG 213 EU402A (vp)mUfGmAfGmAfUmUfUmUfCmAfCmUfUmUfGmA(ps)fG(ps)mC UGAGAUUUUCACUUUGAGC 214 EU402B [ST23 (ps) ] 3ST43 (ps) mGmCmUmCmAmAfAfGfUmGmAmAmAmAmUmCmU (ps) mC (ps) mC GCUCAAAGUGAAAAUCUCC 215 EU403A (vp) mUfCmGfAmGfAmGfUmCfAmAfAmCfUmCfAmU (ps) fA (ps) mA UCGAGAGUCAAACUCAUAA 216 EU403B [ST23(ps) ] 3ST43 (ps) mUmUmAmUmGmAfGfUfUmUmGmAmCmUmCmUmC (ps) mG (ps) mG UUAUGAGUUUGACUCUCGG 217 EU404A (vp) mUfUmCfAmUfAmAfCmAfAmCfAmAfAmAfCmU (ps) fC (ps) mC UUCAUAACAACAAAACUCC 218 EU404B [ST23(ps) ] 3ST43 (ps) mGmGmAmGmUmUfUfUfGmUmUmGmUmUmAmUmG (ps) mA (ps) mC GGAGUUUUGUUGUUAUGAC 219 EU405A (vp) mUfCmGfGmAfCmAfCmGfAmAfGmAfUmGfAmU (ps) fG (ps) mC UCGGACACGAAGAUGAUGC 220 EU405B [ST23 (ps) ] 3ST43 (ps) mGmCmAmUmCmAfUfCfUmUmCmGmUmGmUmCmC (ps) mG (ps) mA GCAUCAUCUUCGUGUCCGA 221 EU406A (vp) mUfUmAfUmCfUmAfCmAfAmUfAmUfUmGfGmA (ps) fC (ps) mU UUAUCUACAAUAUUGGACU 222 EU406B [ST23 (ps) ] 3ST43 (ps) mAmGmUmCmCmAfAfUfAmUmUmGmUmAmGmAmU (ps) mA (ps) mU AGUCCAAUAUUGUAGAUAU 223 EU407A (vp) mUfAmUfAmUfCmUfAmCfAmAfUmAfUmUfGmG (ps) fA (ps) mC UAUAUCUACAAUAUUGGAC 224 EU407B [ST23 (ps) ] 3ST43 (ps) mGmUmCmCmAmAfUfAfUmUmGmUmAmGmAmUmA (ps) mU (ps) mU GUCCAAUAUUGUAGAUAUU 225 EU408A (vp) mUfAmGfAmAfGmCfGmAfGmUfGmAfUmAfGmU (ps) fC (ps) mU UAGAAGCGAGUGAUAGUCU 226 EU408B [ST23 (ps) ] 3ST43 (ps) mAmGmAmCmUmAfUfCfAmCmUmCmGmCmUmUmC (ps) mU (ps) mA AGACUAUCACUCGCUUCUA 227 EU409A (vp) mUfGmGfAmCfUmAfAmCfAmGfGmUfUmCfCmC (ps) fU (ps) mC UGGACUAACAGGUUCCCUC 228 EU409B [ST23 (ps) ] 3ST43 (ps) mGmAmGmGmGmAfAfCfCmUmGmUmUmAmGmUmC (ps) mC (ps) mA GAGGGAACCUGUUAGUCCA 229 EU410A (vp) mUfUmAfAmAfGmUfUmCfCmUfUmAfAmCfCmC (ps) fU (ps) mG UUAAAGUUCCUUAACCCUG 230 EU410B [ST23 (ps) ] 3ST43 (ps) mCmAmGmGmGmUfUfAfAmGmGmAmAmCmUmUmU (ps) mA (ps) mU CAGGGUUAAGGAACUUUAU 231 EU411A (vp) mUfAmCfAmUfAmAfAmGfUmUfCmCfUmUfAmA (ps) fC (ps) mC UACAUAAAGUUCCUUAACC 232 EU411B [ST23 (ps) ] 3ST43 (ps) mGmGmUmUmAmAfGfGfAmAmCmUmUmUmAmUmG (ps) mU (ps) mA GGUUAAGGAACUUUAUGUA 233 EU412A (vp) mUfUmUfAmUfCmAfUmUfGmUfUmUfUmGfUmG (ps) fA (ps) mC UUUAUCAUUGUUUUGUGAC 234 EU412B [ST23 (ps) ] 3ST43 (ps) mGmUmCmAmCmAfAfAfAmCmAmAmUmGmAmUmA (ps) mA (ps) mU GUCACAAAACAAUGAUAAU 235 EU413A (vp) mUfUmAfAmCfAmAfCmAfAmAfAmCfUmCfCmC (ps) fC (ps) mG UUAACAACAAAACUCCCCG 236 EU413B [ST23 (ps) ] 3ST43 (ps) mCmGmGmGmGmAfGfUfUmUmUmGmUmUmGmUmU (ps) mA (ps) mU CGGGGAGUUUUGUUGUUAU 237 EU414A (vp) mUfGmAfCmAfAmAfAmGfAmCfAmCfAmAfGmA (ps) fG (ps) mU UGACAAAAGACACAAGAGU 238 EU414B [ST23 (ps) ] 3ST43 (ps) mAmCmUmCmUmUfGfUfGmUmCmUmUmUmUmGmU (ps) mC (ps) mA ACUCUUGUGUCUUUUGUCA 239 EU201Aun CCUAGGAUCACGCUCUGCU CCUAGGAUCACGCUCUGCU 240 EU201Bun AGCAGAGCGUGAUCCUAGG AGCAGAGCGUGAUCCUAGG 241 EU202Aun GACUUGUUGCAGCUCGCCA GACUUGUUGCAGCUCGCCA 242 EU202Bun UGGCGAGCUGCAACAAGUC UGGCGAGCUGCAACAAGUC 243 EU203Aun CACGACUUGUUGCAGCUCG CACGACUUGUUGCAGCUCG 244 EU203Bun CGAGCUGCAACAAGUCGUG CGAGCUGCAACAAGUCGUG 245 EU204Aun GACACGAAGAUGAUGCCAU GACACGAAGAUGAUGCCAU 246 EU204Bun AUGGCAUCAUCUUCGUGUC AUGGCAUCAUCUUCGUGUC 247 EU205Aun CGGUGAAGGAGAUCAGGUU CGGUGAAGGAGAUCAGGUU 248 EU205Bun AACCUGAUCUCCUUCACCG AACCUGAUCUCCUUCACCG 249 EU209Aun GACCCGAUGAGGUUGUCUA GACCCGAUGAGGUUGUCUA 250 EU209Bun UAGACAACCUCAUCGGGUC UAGACAACCUCAUCGGGUC 251 EU210Aun GGACCCGAUGAGGUUGUCU GGACCCGAUGAGGUUGUCU 252 EU210Bun AGACAACCUCAUCGGGUCC AGACAACCUCAUCGGGUCC 253 EU211Aun GUAAACAGUGCGAAUCUCC GUAAACAGUGCGAAUCUCC 254 EU211Bun GGAGAUUCGCACUGUUUAC GGAGAUUCGCACUGUUUAC 255 EU212Aun GUUGUAAACAGUGCGAAUC GUUGUAAACAGUGCGAAUC 256 EU212Bun GAUUCGCACUGUUUACAAC GAUUCGCACUGUUUACAAC 257 EU213Aun CAUUAUAGGGCUCUGUCAC CAUUAUAGGGCUCUGUCAC 258 EU213Bun GUGACAGAGCCCUAUAAUG GUGACAGAGCCCUAUAAUG 259 EU214Aun GUCAUUAUAGGGCUCCGUC GUCAUUAUAGGGCUCCGUC 260 EU214Bun GACGGAGCCCUAUAAUGAC GACGGAGCCCUAUAAUGAC 261 EU215Aun GAUCAUAUUGAGCUCCUCU GAUCAUAUUGAGCUCCUCU 262 EU215Bun AGAGGAGCUCAAUAUGAUC AGAGGAGCUCAAUAUGAUC 263 EU216Aun GCUGGGUCAUGAUAUCCUC GCUGGGUCAUGAUAUCCUC 264 EU216Bun GAGGAUAUCAUGACCCAGC GAGGAUAUCAUGACCCAGC 265 EU217Aun CUGCGGAUCAUGAAGCAGU CUGCGGAUCAUGAAGCAGU 266 EU217Bun ACUGCUUCAUGAUCCGCAG ACUGCUUCAUGAUCCGCAG 267 EU218Aun GGAGAUUUUCACUUUGAGC GGAGAUUUUCACUUUGAGC 268 EU218Bun GCUCAAAGUGAAAAUCUCC GCUCAAAGUGAAAAUCUCC 269 EU219Aun CUUAUUUCGGGUGUACAGG CUUAUUUCGGGUGUACAGG 270 EU219Bun CCUGUACACCCGAAAUAAG CCUGUACACCCGAAAUAAG 271 EU220Aun GCGCCCGUCUCAAACUUCA GCGCCCGUCUCAAACUUCA 272 EU220Bun UGAAGUUUGAGACGGGCGC UGAAGUUUGAGACGGGCGC 273 EU221Aun CUUAACCCUGUCUUCCCUU CUUAACCCUGUCUUCCCUU 274 EU221Bun AAGGGAAGACAGGGUUAAG AAGGGAAGACAGGGUUAAG 275 EU222Aun CAGUUCCAGGGUAUGGCUC CAGUUCCAGGGUAUGGCUC 276 EU222Bun GAGCCAUACCCUGGAACUG GAGCCAUACCCUGGAACUG 277 EU223Aun CCGAGAGUCAAACUCAUAA CCGAGAGUCAAACUCAUAA 278 EU223Bun UUAUGAGUUUGACUCUCGG UUAUGAGUUUGACUCUCGG 279 EU224Aun GUCAUAACAACAAAACUCC GUCAUAACAACAAAACUCC 280 EU224Bun GGAGUUUUGUUGUUAUGAC GGAGUUUUGUUGUUAUGAC 281 EU225Aun CCAUAUAUCGUUUAGCAAA CCAUAUAUCGUUUAGCAAA 282 EU225Bun UUUGCUAAACGAUAUAUGG UUUGCUAAACGAUAUAUGG 283 EU226Aun AGAUGAUGCCAUCCGGGUU AGAUGAUGCCAUCCGGGUU 284 EU226Bun AACCCGGAUGGCAUCAUCU AACCCGGAUGGCAUCAUCU 285 EU227Aun ACGAAGAUGAUGCCAUCCG ACGAAGAUGAUGCCAUCCG 286 EU227Bun CGGAUGGCAUCAUCUUCGU CGGAUGGCAUCAUCUUCGU 287 EU228Aun UCGGACACGAAGAUGAUGC UCGGACACGAAGAUGAUGC 288 EU228Bun GCAUCAUCUUCGUGUCCGA GCAUCAUCUUCGUGUCCGA 289 EU230Aun UCGGUGAAGGAGAUCAGGU UCGGUGAAGGAGAUCAGGU 290 EU230Bun ACCUGAUCUCCUUCACCGA ACCUGAUCUCCUUCACCGA 291 EU231Aun ACCUCGGUGAAGGAGAUCA ACCUCGGUGAAGGAGAUCA 292 EU231Bun UGAUCUCCUUCACCGAGGU UGAUCUCCUUCACCGAGGU 293 EU233Aun UACAGCUUCAUGCUCUCGC UACAGCUUCAUGCUCUCGC 294 EU233Bun GCGAGAGCAUGAAGCUGUA GCGAGAGCAUGAAGCUGUA 295 EU234Aun AUGAAGAGCAGUGAGUCCU AUGAAGAGCAGUGAGUCCU 296 EU234Bun AGGACUCACUGCUCUUCAU AGGACUCACUGCUCUUCAU 297 EU235Aun AGGUAGUUGCCCUUGCGCC AGGUAGUUGCCCUUGCGCC 298 EU235Bun GGCGCAAGGGCAACUACCU GGCGCAAGGGCAACUACCU 299 EU236Aun UGUGAGGGAGGUGUUGACC UGUGAGGGAGGUGUUGACC 300 EU236Bun GGUCAACACCUCCCUCACA GGUCAACACCUCCCUCACA 301 EU237Aun AUUGUGAGGGAGGUGUUGA AUUGUGAGGGAGGUGUUGA 302 EU237Bun UCAACACCUCCCUCACAAU UCAACACCUCCCUCACAAU 303 EU238Aun UCUAGAAGGAUUGUGAGGG UCUAGAAGGAUUGUGAGGG 304 EU238Bun CCCUCACAAUCCUUCUAGA CCCUCACAAUCCUUCUAGA 305 EU239Aun UUGUCUAGAAGGAUUGUGA UUGUCUAGAAGGAUUGUGA 306 EU239Bun UCACAAUCCUUCUAGACAA UCACAAUCCUUCUAGACAA 307 EU240Aun UGAGGUUGUCUAGAAGGAU UGAGGUUGUCUAGAAGGAU 308 EU240Bun AUCCUUCUAGACAACCUCA AUCCUUCUAGACAACCUCA 309 EU241Aun AUCUCCCCAAAGAUGACAA AUCUCCCCAAAGAUGACAA 310 EU241Bun UUGUCAUCUUUGGGGAGAU UUGUCAUCUUUGGGGAGAU 311 EU243Aun AGCAUAAAGAAUUUGGUGA AGCAUAAAGAAUUUGGUGA 312 EU243Bun UCACCAAAUUCUUUAUGCU UCACCAAAUUCUUUAUGCU 313 EU245Aun ACAGUUUUGGUCCUUAGUU ACAGUUUUGGUCCUUAGUU 314 EU245Bun AACUAAGGACCAAAACUGU AACUAAGGACCAAAACUGU 315 EU246Aun AUAUCCUCUACAGUUUUGG AUAUCCUCUACAGUUUUGG 316 EU246Bun CCAAAACUGUAGAGGAUAU CCAAAACUGUAGAGGAUAU 317 EU247Aun AUAUCUACAAUAUUGGACU AUAUCUACAAUAUUGGACU 318 EU247Bun AGUCCAAUAUUGUAGAUAU AGUCCAAUAUUGUAGAUAU 319 EU248Aun AAUAUCUACAAUAUUGGAC AAUAUCUACAAUAUUGGAC 320 EU248Bun GUCCAAUAUUGUAGAUAUU GUCCAAUAUUGUAGAUAUU 321 EU249Aun UUGACGUAGAGAAUAUCUA UUGACGUAGAGAAUAUCUA 322 EU249Bun UAGAUAUUCUCUACGUCAA UAGAUAUUCUCUACGUCAA 323 EU250Aun AAGUCUUUGACGUAGAGAA AAGUCUUUGACGUAGAGAA 324 EU250Bun UUCUCUACGUCAAAGACUU UUCUCUACGUCAAAGACUU 325 EU251Aun UAGAAGCGAGUGAUAGUCU UAGAAGCGAGUGAUAGUCU 326 EU251Bun AGACUAUCACUCGCUUCUA AGACUAUCACUCGCUUCUA 327 EU252Aun UUGAAGGCAGAGAAGUCAC UUGAAGGCAGAGAAGUCAC 328 EU252Bun GUGACUUCUCUGCCUUCAA GUGACUUCUCUGCCUUCAA 329 EU253Aun UUUCACUUUGAGCUCAUUG UUUCACUUUGAGCUCAUUG 330 EU253Bun CAAUGAGCUCAAAGUGAAA CAAUGAGCUCAAAGUGAAA 331 EU254Aun AACUGGUUGCUGCUGCCUG AACUGGUUGCUGCUGCCUG 332 EU254Bun CAGGCAGCAGCAACCAGUU CAGGCAGCAGCAACCAGUU 333 EU255Aun UUGAGAAGAGUUGUGGUCU UUGAGAAGAGUUGUGGUCU 334 EU255Bun AGACCACAACUCUUCUCAA AGACCACAACUCUUCUCAA 335 EU256Aun UUCAUGUGGGCCCACUGGG UUCAUGUGGGCCCACUGGG 336 EU256Bun CCCAGUGGGCCCACAUGAA CCCAGUGGGCCCACAUGAA 337 EU257Aun UCUUCAUGUGGGCCCACUG UCUUCAUGUGGGCCCACUG 338 EU257Bun CAGUGGGCCCACAUGAAGA CAGUGGGCCCACAUGAAGA 339 EU258Aun UGGACUAACAGGUUCCCUC UGGACUAACAGGUUCCCUC 340 EU258Bun GAGGGAACCUGUUAGUCCA GAGGGAACCUGUUAGUCCA 341 EU259Aun UUCCUUAACCCUGUCUUCC UUCCUUAACCCUGUCUUCC 342 EU259Bun GGAAGACAGGGUUAAGGAA GGAAGACAGGGUUAAGGAA 343 EU260Aun AGUUCCUUAACCCUGUCUU AGUUCCUUAACCCUGUCUU 344 EU260Bun AAGACAGGGUUAAGGAACU AAGACAGGGUUAAGGAACU 345 EU261Aun AUAAAGUUCCUUAACCCUG AUAAAGUUCCUUAACCCUG 346 EU261Bun CAGGGUUAAGGAACUUUAU CAGGGUUAAGGAACUUUAU 347 EU262Aun AAAUAAAGUUCCUUAACCC AAAUAAAGUUCCUUAACCC 348 EU262Bun GGGUUAAGGAACUUUAUUU GGGUUAAGGAACUUUAUUU 349 EU263Aun UACAUAAAGUUCCUUAACC UACAUAAAGUUCCUUAACC 350 EU263Bun GGUUAAGGAACUUUAUGUA GGUUAAGGAACUUUAUGUA 351 EU264Aun AUUAUCAUGUCCCUGAUCA AUUAUCAUGUCCCUGAUCA 352 EU264Bun UGAUCAGGGACAUGAUAAU UGAUCAGGGACAUGAUAAU 353 EU265Aun AUAAAGAACCUGCUUCCGU AUAAAGAACCUGCUUCCGU 354 EU265Bun ACGGAAGCAGGUUCUUUAU ACGGAAGCAGGUUCUUUAU 355 EU266Aun ACUAACUUCAGCAAGGGCA ACUAACUUCAGCAAGGGCA 356 EU266Bun UGCCCUUGCUGAAGUUAGU UGCCCUUGCUGAAGUUAGU 357 EU267Aun UGGUGCAAGACAAUCCCUG UGGUGCAAGACAAUCCCUG 358 EU267Bun CAGGGAUUGUCUUGCACCA CAGGGAUUGUCUUGCACCA 359 EU269Aun UUGGAAAUAAUGAUGACCC UUGGAAAUAAUGAUGACCC 360 EU269Bun GGGUCAUCAUUAUUUCCAA GGGUCAUCAUUAUUUCCAA 361 EU271Aun AUUAUCAUUGUUUUGUGAC AUUAUCAUUGUUUUGUGAC 362 EU271Bun GUCACAAAACAAUGAUAAU GUCACAAAACAAUGAUAAU 363 EU273Aun UCCCCGAGAGUCAAACUCA UCCCCGAGAGUCAAACUCA 364 EU273Bun UGAGUUUGACUCUCGGGGA UGAGUUUGACUCUCGGGGA 365 EU274Aun AACUCCCCGAGAGUCAAAC AACUCCCCGAGAGUCAAAC 366 EU274Bun GUUUGACUCUCGGGGAGUU GUUUGACUCUCGGGGAGUU 367 EU275Aun AACAACAAAACUCCCCGAG AACAACAAAACUCCCCGAG 368 EU275Bun CUCGGGGAGUUUUGUUGUU CUCGGGGAGUUUUGUUGUU 369 EU276Aun AUAACAACAAAACUCCCCG AUAACAACAAAACUCCCCG 370 EU276Bun CGGGGAGUUUUGUUGUUAU CGGGGAGUUUUGUUGUUAU 371 EU277Aun UGACAAAAGACACAAGAGU UGACAAAAGACACAAGAGU 372 EU277Bun ACUCUUGUGUCUUUUGUCA ACUCUUGUGUCUUUUGUCA 373 EU279Aun UCUAGGAUCACGCUCUGCU UCUAGGAUCACGCUCUGCU 374 EU279Bun AGCAGAGCGUGAUCCUAGA AGCAGAGCGUGAUCCUAGA 375 EU280Aun UACUUGUUGCAGCUCGCCA UACUUGUUGCAGCUCGCCA 376 EU280Bun UGGCGAGCUGCAACAAGUA UGGCGAGCUGCAACAAGUA 377 EU281Aun UACGACUUGUUGCAGCUCG UACGACUUGUUGCAGCUCG 378 EU281Bun CGAGCUGCAACAAGUCGUA CGAGCUGCAACAAGUCGUA 379 EU282Aun UACACGAAGAUGAUGCCAU UACACGAAGAUGAUGCCAU 380 EU282Bun AUGGCAUCAUCUUCGUGUA AUGGCAUCAUCUUCGUGUA 381 EU283Aun UGGUGAAGGAGAUCAGGUU UGGUGAAGGAGAUCAGGUU 382 EU283Bun AACCUGAUCUCCUUCACCA AACCUGAUCUCCUUCACCA 383 EU284Aun UUCACGUUGACCAGCUGCU UUCACGUUGACCAGCUGCU 384 EU284Bun AGCAGCUGGUCAACGUGAA AGCAGCUGGUCAACGUGAA 385 EU287Aun UACCCGAUGAGGUUGUCUA UACCCGAUGAGGUUGUCUA 386 EU287Bun UAGACAACCUCAUCGGGUA UAGACAACCUCAUCGGGUA 387 EU289Aun UUAAACAGUGCGAAUCUCC UUAAACAGUGCGAAUCUCC 388 EU289Bun GGAGAUUCGCACUGUUUAA GGAGAUUCGCACUGUUUAA 389 EU290Aun UUUGUAAACAGUGCGAAUC UUUGUAAACAGUGCGAAUC 390 EU290Bun GAUUCGCACUGUUUACAAA GAUUCGCACUGUUUACAAA 391 EU291Aun UAUUAUAGGGCUCUGUCAC UAUUAUAGGGCUCUGUCAC 392 EU291Bun GUGACAGAGCCCUAUAAUA GUGACAGAGCCCUAUAAUA 393 EU292Aun UUCAUUAUAGGGCUCCGUC UUCAUUAUAGGGCUCCGUC 394 EU292Bun GACGGAGCCCUAUAAUGAA GACGGAGCCCUAUAAUGAA 395 EU293Aun UAUCAUAUUGAGCUCCUCU UAUCAUAUUGAGCUCCUCU 396 EU293Bun AGAGGAGCUCAAUAUGAUA AGAGGAGCUCAAUAUGAUA 397 EU294Aun UCUGGGUCAUGAUAUCCUC UCUGGGUCAUGAUAUCCUC 398 EU294Bun GAGGAUAUCAUGACCCAGA GAGGAUAUCAUGACCCAGA 399 EU295Aun UUGCGGAUCAUGAAGCAGU UUGCGGAUCAUGAAGCAGU 400 EU295Bun ACUGCUUCAUGAUCCGCAA ACUGCUUCAUGAUCCGCAA 401 EU296Aun UGAGAUUUUCACUUUGAGC UGAGAUUUUCACUUUGAGC 402 EU296Bun GCUCAAAGUGAAAAUCUCA GCUCAAAGUGAAAAUCUCA 403 EU297Aun UUUAUUUCGGGUGUACAGG UUUAUUUCGGGUGUACAGG 404 EU297Bun CCUGUACACCCGAAAUAAA CCUGUACACCCGAAAUAAA 405 EU298Aun UCGCCCGUCUCAAACUUCA UCGCCCGUCUCAAACUUCA 406 EU298Bun UGAAGUUUGAGACGGGCGA UGAAGUUUGAGACGGGCGA 407 EU299Aun UUUAACCCUGUCUUCCCUU UUUAACCCUGUCUUCCCUU 408 EU299Bun AAGGGAAGACAGGGUUAAA AAGGGAAGACAGGGUUAAA 409 EU300Aun UAGUUCCAGGGUAUGGCUC UAGUUCCAGGGUAUGGCUC 410 EU300Bun GAGCCAUACCCUGGAACUA GAGCCAUACCCUGGAACUA 411 EU301Aun UCGAGAGUCAAACUCAUAA UCGAGAGUCAAACUCAUAA 412 EU301Bun UUAUGAGUUUGACUCUCGA UUAUGAGUUUGACUCUCGA 413 EU302Aun UUCAUAACAACAAAACUCC UUCAUAACAACAAAACUCC 414 EU302Bun GGAGUUUUGUUGUUAUGAA GGAGUUUUGUUGUUAUGAA 415 EU303Aun UCAUAUAUCGUUUAGCAAA UCAUAUAUCGUUUAGCAAA 416 EU303Bun UUUGCUAAACGAUAUAUGA UUUGCUAAACGAUAUAUGA 417 EU406Aun UUAUCUACAAUAUUGGACU UUAUCUACAAUAUUGGACU 418 EU407Aun UAUAUCUACAAUAUUGGAC UAUAUCUACAAUAUUGGAC 419 EU410Aun UUAAAGUUCCUUAACCCUG UUAAAGUUCCUUAACCCUG 420 EU412Aun UUUAUCAUUGUUUUGUGAC UUUAUCAUUGUUUUGUGAC 421 EU413Aun UUAACAACAAAACUCCCCG UUAACAACAAAACUCCCCG 422 Mm n1 A GAACUGAGAAGGAGAGAAA GAACUGAGAAGGAGAGAAA 423 Mm n1 B UUUCUCUCCUUCUCAGUUC UUUCUCUCCUUCUCAGUUC 424 Mm n2 A GGGAGAAGCUGAUGGAGAU GGGAGAAGCUGAUGGAGAU 425 Mm n2 B AUCUCCAUCAGCUUCUCCC AUCUCCAUCAGCUUCUCCC 426 Mm n3 A CAAUGAACUCAAAGUGAAA CAAUGAACUCAAAGUGAAA 427 Mm n3 B UUUCACUUUGAGUUCAUUG UUUCACUUUGAGUUCAUUG 428 Mm n4 A CGGGAGAAGCUGAUGGAGA CGGGAGAAGCUGAUGGAGA 429 Mm n4 B UCUCCAUCAGCUUCUCCCG UCUCCAUCAGCUUCUCCCG 430 Mm n5 A UGGUGAAGGAGGAGUUAAA UGGUGAAGGAGGAGUUAAA 431 Mm n5 B UUUAACUCCUCCUUCACCA UUUAACUCCUCCUUCACCA 432 Mm n6 A GUGAAGGAGGAGUUAAAUA GUGAAGGAGGAGUUAAAUA 433 Mn n6 B UAUUUAACUCCUCCUUCAC UAUUUAACUCCUCCUUCAC 434 Hs n1 A GAUUGUAGCUGUUAAGAAA GAUUGUAGCUGUUAAGAAA 435 Hs n1 B UUUCUUAACAGCUACAAUC UUUCUUAACAGCUACAAUC 436 Hs n2 A AGGCAGAGCUCAAGGGAGA AGGCAGAGCUCAAGGGAGA 437 Hs n2 B UCUCCCUUGAGCUCUGCCU UCUCCCUUGAGCUCUGCCU 438 Hs n3 A GAUUGUAGCUGUUAAGAAA GAUUGUAGCUGUUAAGAAA 439 Hs n3 B UUUCUUAACAGCUACAAUC UUUCUUAACAGCUACAAUC 440 Hs n4 A CGAUGGAGAUUUAGAGUAU CGAUGGAGAUUUAGAGUAU 441 Hs n4 B AUACUCUAAAUCUCCAUCG AUACUCUAAAUCUCCAUCG 442 Hs n5 A GCUGAUGAGUGCAAAGAAA GCUGAUGAGUGCAAAGAAA 443 Hs n5 B UUUCUUUGCACUCAUCAGC UUUCUUUGCACUCAUCAGC 444 Hs n6 A CCUCAAAGCUCAAGGCACA CCUCAAAGCUCAAGGCACA 445 Hs n6 B UGUGCCUUGAGCUUUGAGG UGUGCCUUGAGCUUUGAGG 446 Hs n7 A UCCAUUAUCUCCGACAUGG UCCAUUAUCUCCGACAUGG 447 Hs n7 B CCAUGUCGGAGAUAAUGGA CCAUGUCGGAGAUAAUGGA 448 Hs n7 Ao UCCAUUAUCUCCGACAUGGTG UCCAUUAUCUCCGACAUGG TG 449 Hs n7 Bo CCAUGUCGGAGAUAAUGGATT CCAUGUCGGAGAUAAUGGA TT 450 Mm n7 A UUUGGUGAGAACAAUAGUG UUUGGUGAGAACAAUAGUG 451 Mm n7 B CACUAUUGUUCUCACCAAA CACUAUUGUUCUCACCAAA 452 Mm n7 Ao UUUGGUGAGAACAAUAGUG UUUGGUGAGAACAAUAGUG TT 453 Mm n7 Bo CACUAUUGUUCUCACCAAA CACUAUUGUUCUCACCAAA TT 454 CNNM4 shRNA UCUCUGCCUUCAAGGAUGCGGACAAUGAG UCUCUGCCUUCAAGGAUGC GGACAAUGAG 455 EU415B [ST23 (ps) ] 3ST41 (ps) mGmGmAmGmUmUfUfUfGmUmUmGmUmUmAmUmG (ps) mA (ps) mC GGAGUUUUGUUGUUAUGAC 456 EU416A mG (ps) fU (ps) mCfAmUfAmLAfCmLAfAmCfAmLAfAmAfCmU (ps) fC (ps) mC GUCAUAACAACAAAACUCC 457 EU418A (vp) mUfUmCfAmUfAmAfCmAfAmCfAmAfAmAfCmUfC (ps2) mC UUCAUAACAACAAAACUCC 458 EU418B [ST23 ] 3ST41mG (ps2) mGmAmGmUmUfUfUfGmUmUmGmUmUmAmUmGmA (ps2) mC GGAGUUUUGUUGUUAUGAC 459 EU419B [ST23 ] 3ST41mG (ps2) mGmAmGmUmUfUfUfGmUmUmGmUmUmAmUmGmA (ps2) mA GGAGUUUUGUUGUUAUGAA 460 EU420B [ST23 (ps) ] 3ST41 (ps) mAmCmUmCmUmUfGfUfGmUmCmUmUmUmUmGmU (ps) mC (ps) mA ACUCUUGUGUCUUUUGUCA 461 EU422A (vp) mUfGmAfCmAfAmAfAmGfAmCfAmCfAmAfGmA fG (ps2) mU UGACAAAAGACACAAGAGU 462 EU422B [ST23 ] 3ST41mA (ps2) mCmUmCmUmUfGfUfGmUmCmUmUmUmUmGmUmC (ps2) mA ACUCUUGUGUCUUUUGUCA 463 EU423A mU (ps) fC (ps) mGfAmAfGmUfAmUfUmCfCmGfCmGfUmA (ps) fC (ps) mG UCGAAGUAUUCCGCGUACG 464 EU423B [ST23 (ps) ] 3ST41 (ps) fCmGfUmAfCmGfCmGfGmAfAmUfAmCfUmUfC (ps) mG(ps) fA CGUACGCGGAAUACUUCGA 465 EU304A mU (ps) fC (ps) mCfUmUfGmUfCmCfGmUfCmCfAmCfUmU (ps) fC (ps) mA UCCUUGUCCGUCCACUUCA 466 EU304B mU (ps) mG (ps) mAmAmGmUfGfGfAmCmGmGmAmCmAmAmG (ps) mG (ps) mA UGAAGUGGACGGACAAGGA 467 EU305A mC (ps) fA (ps) mAfUmCfUmUfGmCfGmGfGmCfAmUfArnG (ps) fC (ps) mG CAAUCUUGCGGGCAUAGCG 468 EU305B mC (ps) mG (ps) mCmUmAmUfGfCfCmCmGmCmAmAmGmAmU (ps) mU (ps) mG CGCUAUGCCCGCAAGAUUG 469 EU306A mU (ps) fA (ps) mAfAmCfAmGfUmGfCmGfAmAfUmCfUmC (ps) fC (ps) mU UAAACAGUGCGAAUCUCCU 470 EU306B mA (ps) mG (ps) mGmAmGmAfUfUfCmGmCmAmCmUmGmUmU (ps) mU (ps) mA AGGAGAUUCGCACUGUUUA 471 EU307A mA (ps) fU (ps) mCfAmUfAmUfUmGfAmGfCmUfCmCfUmC (ps) fU (ps) mU AUCAUAUUGAGCUCCUCUU 472 EU307B mA (ps) mA (ps) mGmAmGmGfAfGfCmUmCmAmAmUmAmUmG (ps) mA (ps) mU AAGAGGAGCUCAAUAUGAU 473 EU308A mU (ps) fU (ps) mGfUmUfCmUfUmCfUmCfAmGfAmCfArnC (ps) fC (ps) mC UUGUUCUUCUCAGACACCC 474 EU308B mG (ps) mG (ps) mGmUmGmUfCfUfGmAmGmAmAmGmAmAmC (ps) mA (ps) mA GGGUGUCUGAGAAGAACAA 475 EU309A mG (ps) fA (ps) mGfAmUfUmUfUmCfAmCfUmUfUmGfAmG (ps) fC (ps) mU GAGAUUUUCACUUUGAGCU 476 EU309B mA (ps) mG (ps) mCmUmCmAfAfAfGmUmGmAmAmAmAmUmC (ps) mU (ps) mC AGCUCAAAGUGAAAAUCUC 477 EU310A mA (ps) fA (ps) mUfGmAfCmAfUmCfUmGfGmGfUmAfCmU (ps) fU (ps) mG AAUGACAUCUGGGUACUUG 478 EU310B mC (ps) mA (ps) mAmGmUmAfCfCfCmAmGmAmUmGmUmCmA (ps) mU (ps) mU CAAGUACCCAGAUGUCAUU 479 EU311A mU (ps) fU (ps) mAfUmUfUmCfGmGfGmUfGmUfAmCfAmG (ps) fG (ps) mU UUAUUUCGGGUGUACAGGU 480 EU311B mA (ps) mC (ps) mCmUmGmUfAfCfAmCmCmCmGmAmAmAmU (ps) mA (ps) mA ACCUGUACACCCGAAAUAA 481 EU312A mU (ps) fU (ps) mGfAmUfGmUfAmCfUmGfCmAfAmGfUmC (ps) fC (ps) mA UUGAUGUACUGCAAGUCCA 482 EU312B mU (ps) mG (ps) mGmAmCmUfUfGfCmAmGmUmAmCmAmUmC (ps) mA (ps) mA UGGACUUGCAGUACAUCAA 483 EU313A mG (ps) fA (ps) mCfUmUfCmUfCmGfGmCfCmAfAmGfUmU (ps) fC (ps) mU GACUUCUCGGCCAAGUUCU 484 EU313B mA (ps) mG (ps) mAmAmCmUfUfGfGmCmCmGmAmGmAmAmG (ps) mU (ps) mC AGAACUUGGCCGAGAAGUC 485 EU314A mA (ps) fA (ps) mCfAmGfGmUfUmCfCmCfUmCfUmCfUmU (ps) fC (ps) mA AACAGGUUCCCUCUCUUCA 486 EU314B mU (ps) mG (ps) mAmAmGmAfGfAfGmGmGmAmAmCmCmUmG (ps) mU (ps) mU UGAAGAGAGGGAACCUGUU 487 EU315A mA (ps) fC (ps) mAfUmAfAmAfGmAfAmCfCmUfGmCfUmU (ps) fC (ps) mC ACAUAAAGAACCUGCUUCC 488 EU315B mG (ps) mG (ps) mAmAmGmCfAfGfGmUmUmCmUmUmUmAmU (ps) mG (ps) mU GGAAGCAGGUUCUUUAUGU 489 EU316A mU(ps)fA (ps) mCfUmAfCmUfAmAfCmUfUmCfAmGfCmA (ps) fA (ps) mG UACUACUAACUUCAGCAAG 490 EU316B mC(ps)mU(ps)mUmGmCmUfGfAfAmGmUmUmAmGmUmAmG(ps)mU(ps)mA CUUGCUGAAGUUAGUAGUA 491 EU317A mA (ps) fG (ps) mUfAmCfUmAfCmUfAmAfCmUfUmCfAmG (ps) fC (ps) mA AGUACUACUAACUUCAGCA 492 EU317B mU(ps)mG(ps)mCmUmGmAfAfGfUmUmAmGmUmAmGmUmA(ps)mC(ps)mU UGCUGAAGUUAGUAGUACU 493 EU318A mA(ps)fG(ps)mUfUmCfUmAfUmGfGmCfUmAfGmGfAmG(ps)fA(ps)mC AGUUCUAUGGCUAGGAGAC 494 EU318B mG(ps)mU(ps)mCmUmCmCfUfAfGmCmCmAmUmAmGmAmA(ps)mC(ps)mU GUCUCCUAGCCAUAGAACU 495 EU319A mA(ps)fA(ps)mCfAmAfAmAfGmUfCmUfGmGfUmGfUmC(ps)fU(ps)mU AACAAAAGUCUGGUGUCUU 496 EU319B mA(ps)mA(ps)mGmAmCmAfCfCfAmGmAmCmUmUmUmUmG(ps)mU(ps)mU AAGACACCAGACUUUUGUU 497 EU320A mG(ps)fA(ps)mAfAmUfAmAfUmGfAmUfGmAfCmCfCmU(ps)fC(ps)mU GAAAUAAUGAUGACCCUCU 498 EU320B mA(ps)mG(ps)mAmGmGmGfUfCfAmUmCmAmUmUmAmUmU(ps)mU(ps)mC AGAGGGUCAUCAUUAUUUC 499 EU321A mA(ps)fA(ps)mAfAmUfAmAfAmUfAmCfGmGfCmGfGmC(ps)fU(ps)mG AAAAUAAAUACGGCGGCUG 500 EU321B mC(ps)mA(ps)mGmCmCmGfCfCfGmUmAmUmUmUmAmUmU(ps)mU(ps)mU CAGCCGCCGUAUUUAUUUU 501 EU322A mA (ps) fU (ps) mGfCmAfAmAfAmUfAmAfAmUfAmCfGmG (ps) fC (ps) mG AUGCAAAAUAAAUACGGCG 502 EU322B mC(ps)mG(ps)mCmCmGmUfAfUfUmUmAmUmUmUmUmGmC(ps)mA(ps)mU CGCCGUAUUUAUUUUGCAU 503 EU323A mA(ps)fG(ps)mUfCmAfAmAfCmUfCmAfUmAfAmCfGmC(ps)fC(ps)mA AGUCAAACUCAUAACGCCA 504 EU323B mU(ps)mG(ps)mGmCmGmUfUfAfUmGmAmGmUmUmUmGmA(ps)mC(ps)mU UGGCGUUAUGAGUUUGACU 505 EU324A mC (ps) fA (ps) mUfAmAfCmAfAmCfAmAfAmAfCmUfCmC (ps) fC (ps) mC CAUAACAACAAAACUCCCC 506 EU324B mG(ps)mG(ps)mGmGmAmGfUfUfUmUmGmUmUmGmUmUmA(ps)mU(ps)mG GGGGAGUUUUGUUGUUAUG 507 EU325A mA(ps)fG(ps)mUfCmAfUmAfAmCfAmAfCmAfAmAfAmC(ps)fU(ps)mC AGUCAUAACAACAAAACUC 508 EU325B mG(ps)mA(ps)mGmUmUmUfUfGfUmUmGmUmUmAmUmGmA(ps)mC(ps)mU GAGUUUUGUUGUUAUGACU 509 EU424A mA(ps)fA(ps)mUfGmAfCmAfUmCfUmGfGmGfUmAfCmU(ps)fU(ps)mG AAUGACAUCUGGGUACUUG 510 EU424B [ST23(ps)]3ST41(ps)mCmAmAmGmUmAfCfCfCmAmGmAmUmGmUmCmA(ps)mU(ps)mU CAAGUACCCAGAUGUCAUU 511 EU425A mA(ps)fA(ps)mCfAmGfGmUfUmCfCmCfUmCfUmCfUmU(ps)fC(ps)mA AACAGGUUCCCUCUCUUCA 512 EU425B [ST23(ps)]3ST41(ps)mUmGmAmAmGmAfGfAfGmGmGmAmAmCmCmUmG(ps)mU(ps)mU UGAAGAGAGGGAACCUGUU 513 EU426A mA (ps) fA (ps) mAfAmUfAmAfAmUfAmCfGmGfCmGfGmC (ps) fU (ps) mG AAAAUAAAUACGGCGGCUG 514 EU426B [ST23(ps)]3ST41(ps)mCmAmGmCmCmGfCfCfGmUmAmUmUmUmAmUmU(ps)mU(ps)mU CAGCCGCCGUAUUUAUUUU 515 EU427A mA (ps) fU (ps) mGfCmAfAmAfAmUfAmAfAmUfAmCfGmG (ps) fC (ps) mG AUGCAAAAUAAAUACGGCG 516 EU427B [ST23 (ps) ] 3ST41 (ps) mCmGmCmCmGmUfAfUfUmUmAmUmUmUmUmGmC (ps) mA (ps) mU CGCCGUAUUUAUUUUGCAU 517 EU428A mA (ps) fG (ps) mUfCmAfUmAfAmCfAmAfCmAfAmAfAmC (ps) fU (ps) mC AGUCAUAACAACAAAACUC 518 EU428B [ST23(ps)]3ST41(ps)mGmAmGmUmUmUfUfGfUmUmGmUmUmAmUmGmA(ps)mC(ps)mU GAGUUUUGUUGUUAUGACU 519 EU429A (vp)mUfAmUfGmAfCmAfUmCfUmGfGmGfUmAfCmU(ps)fU(ps)mG UAUGACAUCUGGGUACUUG 520 EU430A (vp) mUfAmCfAmGfGmUfUmCfCmCfUmCfUmCfUmU (ps) fC (ps) mA UACAGGUUCCCUCUCUUCA 521 EU431A (vp) mUfAmAfAmUfAmAfAmUfAmCfGmGfCmGfGmC (ps) fU (ps) mG UAAAUAAAUACGGCGGCUG 522 EU432A (vp)mUfUmGfCmAfAmAfAmUfAmAfAmUfAmCfGmG(ps)fC(ps)mG UUGCAAAAUAAAUACGGCG 523 EU433A (vp) mUfGmUfCmAfUmAfAmCfAmAfCmAfAmAfAmC (ps) fU (ps) mC UGUCAUAACAACAAAACUC 524 EU304Aun UCCUUGUCCGUCCACUUCA UCCUUGUCCGUCCACUUCA 525 EU304Bun UGAAGUGGACGGACAAGGA UGAAGUGGACGGACAAGGA 526 EU307Aun AUCAUAUUGAGCUCCUCUU AUCAUAUUGAGCUCCUCUU 527 EU307Bun AAGAGGAGCUCAAUAUGAU AAGAGGAGCUCAAUAUGAU 528 EU310Aun AAUGACAUCUGGGUACUUG AAUGACAUCUGGGUACUUG 529 EU310Bun CAAGUACCCAGAUGUCAUU CAAGUACCCAGAUGUCAUU 530 EU312Aun UUGAUGUACUGCAAGUCCA UUGAUGUACUGCAAGUCCA 531 EU312Bun UGGACUUGCAGUACAUCAA UGGACUUGCAGUACAUCAA 532 EU314Aun AACAGGUUCCCUCUCUUCA AACAGGUUCCCUCUCUUCA 533 EU314Bun UGAAGAGAGGGAACCUGUU UGAAGAGAGGGAACCUGUU 534 EU316Aun UACUACUAACUUCAGCAAG UACUACUAACUUCAGCAAG 535 EU316Bun CUUGCUGAAGUUAGUAGUA CUUGCUGAAGUUAGUAGUA 536 EU317Aun AGUACUACUAACUUCAGCA AGUACUACUAACUUCAGCA 537 EU317Bun UGCUGAAGUUAGUAGUACU UGCUGAAGUUAGUAGUACU 538 EU319Aun AACAAAAGUCUGGUGUCUU AACAAAAGUCUGGUGUCUU 539 EU319Bun AAGACACCAGACUUUUGUU AAGACACCAGACUUUUGUU 540 EU321Aun AAAAUAAAUACGGCGGCUG AAAAUAAAUACGGCGGCUG 541 EU321Bun CAGCCGCCGUAUUUAUUUU CAGCCGCCGUAUUUAUUUU 542 EU322Aun AUGCAAAAUAAAUACGGCG AUGCAAAAUAAAUACGGCG 543 EU322Bun CGCCGUAUUUAUUUUGCAU CGCCGUAUUUAUUUUGCAU 544 EU323Aun AGUCAAACUCAUAACGCCA AGUCAAACUCAUAACGCCA 545 EU323Bun UGGCGUUAUGAGUUUGACU UGGCGUUAUGAGUUUGACU 546 EU325Aun AGUCAUAACAACAAAACUC AGUCAUAACAACAAAACUC 547 EU325Bun GAGUUUUGUUGUUAUGACU GAGUUUUGUUGUUAUGACU 548 EU429Aun UAUGACAUCUGGGUACUUG UAUGACAUCUGGGUACUUG 549 EU430Aun UACAGGUUCCCUCUCUUCA UACAGGUUCCCUCUCUUCA 550 EU431Aun UAAAUAAAUACGGCGGCUG UAAAUAAAUACGGCGGCUG 551 EU432Aun UUGCAAAAUAAAUACGGCG UUGCAAAAUAAAUACGGCG 552 EU433Aun UGUCAUAACAACAAAACUC UGUCAUAACAACAAAACUC 553 EU420B without linker mAmCmUmCmUmUfGfUfGmUmCmUmUmUmUmGmU (ps) mC (ps) mA ACUCUUGUGUCUUUUGUCA 554 EU422B without linker mA (ps2) mCmUmCmUmUfGfUfGmUmCmUmUmUmUmGmUmC (ps2) mA ACUCUUGUGUCUUUUGUCA 

1-16. (canceled)
 17. A double-stranded nucleic acid for inhibiting expression of CNNM4, wherein the nucleic acid comprises a first strand and a second strand, wherein the first strand sequence comprises a sequence of at least 15 nucleotides differing by no more than 3 nucleotides from any one of the sequences selected from SEQ ID NO: 371, 243, 267, 277, 279, 287, 317, 319, 325, 333, 345, 347, 349, 361, 367, 369, 377, 401, 411, 413, 415, 420, 421, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 549, 550, 551 and
 552. 18. The nucleic acid of claim 17, wherein the first strand and the second strand form a duplex region of from 17-25 nucleotides in length.
 19. The nucleic acid of claim 17, wherein the nucleic acid mediates RNA interference.
 20. The nucleic acid of claim 17, wherein at least one nucleotide of the first and/or second strand is a modified nucleotide.
 21. The nucleic acid of claim 17, wherein at least nucleotides 2 and 14 of the first strand are modified by a first modification, the nucleotides being numbered consecutively starting with nucleotide number 1 at the 5′ end of the first strand.
 22. The nucleic acid of claim 17, wherein the first strand has a terminal 5′ (E)-vinylphosphonate nucleotide at its 5′ end.
 23. The nucleic acid of claim 17, wherein the nucleic acid comprises a phosphorothioate linkage between the terminal two or three 3′ nucleotides and/or 5′ nucleotides of the first and/or the second strand.
 24. The nucleic acid of claim 17, comprising a phosphorodithioate linkage between each of the two, three or four terminal nucleotides at the 3′ end of the first strand and/or comprising a phosphorodithioate linkage between each of the two, three or four terminal nucleotides at the 3′ end of the second strand and/or a phosphorodithioate linkage between each of the two, three or four terminal nucleotides at the 5′ end of the second strand and comprising a linkage other than a phosphorodithioate linkage between the two, three or four terminal nucleotides at the 5′ end of the first strand.
 25. The nucleic acid of claim 17, wherein the nucleic acid is conjugated to a ligand.
 26. The nucleic acid of claim 25, wherein the ligand comprises (i) one or more N-acetyl galactosamine (GalNAc) moieties or derivatives thereof, and (ii) a linker, wherein the linker conjugates the at least one GalNAc moiety or derivative thereof to the nucleic acid.
 27. A composition comprising a nucleic acid of claim 17 and a solvent and/or a delivery vehicle and/or a physiologically acceptable excipient and/or a carrier and/or a salt and/or a diluent and/or a buffer and/or a preservative and/or a further therapeutic agent selected from the group comprising an oligonucleotide, a small molecule, a monoclonal antibody, a polyclonal antibody and a peptide.
 28. A method of preventing, decreasing the risk of suffering from, or treating a disease, disorder or syndrome comprising administering a pharmaceutically effective amount of the nucleic acid of claim 17 to an individual in need of treatment.
 29. The method of claim 28, wherein the disease, disorder or syndrome is a liver disease, a kidney disease or a lung disease.
 30. The method of claim 28, wherein the disease is a liver disease selected from the group consisting of non-alcoholic steatohepatitis (NASH), liver cirrhosis, hepatocellular carcinoma (HCC), drug-induced liver injury (DILI), non-alcoholic fatty liver disease (NAFLD), fatty liver, liver cancer, liver fibrosis, veno-occlusive liver disease, hepatic sinusoidal obstruction syndrome (SOS), steatosis, Budd-Chiari syndrome, viral hepatitis B, viral hepatitis C, alcoholic hepatitis, hepatic ischemia reperfusion injury, primary biliary cirrhosis (PBC), a chronic liver disease, an acute liver disease, liver damage, a non-proliferative liver disease cholangiocarcinoma (bile duct cancer), a disease associated with hypomagnesemia in the liver.
 31. The method of claim 30, wherein the liver disease is selected from the group consisting of non-alcoholic steatohepatitis (NASH), liver cirrhosis, hepatocellular carcinoma (HCC), drug-induced liver injury (DILI) and non-alcoholic fatty liver disease (NAFLD).
 32. A method of preventing, decreasing the risk of suffering from, or treating a disease, disorder or syndrome comprising administering a pharmaceutically effective amount of the composition of claim 27 to an individual in need of treatment.
 33. The method of claim 32, wherein the disease, disorder or syndrome is a liver disease, a kidney disease or a lung disease.
 34. The method of claim 32, wherein the disease is a liver disease selected from the group consisting of non-alcoholic steatohepatitis (NASH), liver cirrhosis, hepatocellular carcinoma (HCC), drug-induced liver injury (DILI), non-alcoholic fatty liver disease (NAFLD), fatty liver, liver cancer, liver fibrosis, veno-occlusive liver disease, hepatic sinusoidal obstruction syndrome (SOS), steatosis, Budd-Chiari syndrome, viral hepatitis B, viral hepatitis C, alcoholic hepatitis, hepatic ischemia reperfusion injury, primary biliary cirrhosis (PBC), a chronic liver disease, an acute liver disease, liver damage, a non-proliferative liver disease cholangiocarcinoma (bile duct cancer), a disease associated with hypomagnesemia in the liver.
 35. The method of claim 34, wherein the liver disease is selected from the group consisting of non-alcoholic steatohepatitis (NASH), liver cirrhosis, hepatocellular carcinoma (HCC), drug-induced liver injury (DILI) and non-alcoholic fatty liver disease (NAFLD).
 36. The nucleic acid of claim 20, wherein the modified nucleotide is a non-naturally occurring nucleotide such as a 2′-F modified nucleotide. 